Quinazolinone compounds

ABSTRACT

The present invention relates to quinazolinone compounds, processes for their preparation and their use as pharmaceutical agents for the treatment of Parkinson&#39;s disease (PD). The quinazolinone compounds are of general formula (I).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/142,162, filed Aug. 25, 2012, which is the National Phase under§371 of International Application No. PCT/AU2009/001701, filed Dec. 23,2009, which claims the benefit of Australian Patent Application No.2008906650, filed Dec. 24, 2008, the entire contents of which areincorporated herein by reference.

FIELD

The present invention relates to quinazolinone compounds, processes fortheir preparation and their use as pharmaceutical agents for thetreatment of Parkinson's disease (PD).

BACKGROUND

PD is a progressive illness that is symptomatically characterised byslowness of movement (bradykinesia), rigidity and/or tremor and posturalinstability. Patients with PD have a deficiency of the neurotransmitterdopamine, due to chronic and progressive degeneration of the substantianigra in the brain.

The cause of PD is unknown, however the deficiency in dopamine has ledto the widespread use of dopamine-replacing agents as symptomatictreatments for the disease. The most commonly prescribed drug for thistreatment is L-3,4-dihydroxyphenylalanine (L-dopa) and dopamineagonists.

These symptomatic treatments are successful in increasing the quality oflife of patients suffering from PD. Such dopamine-replacement treatmentsdo however have significant limitations, particularly in long-termtreatment. These limitations include fluctuations in the efficacy of thetreatment, leading to the “on-off” phenomenon and appearance ofside-effects which manifest as abnormal involuntary movements.

Other than L-dopa, the most common medicaments for alleviating thesemotor symptoms are dopamine agonists such asrotigotine, pramipexole,bromocriptine, ropinirole, cabergoline, pergolide, apomorphine andlisuride, anticholinergic agents, N-methyl d-aspartate (NMDA)antagonists, beta-blockers as well as the Monoamine oxidase B (MAO-B)inhibitor selegeline and the Catechol-O-methyl transferase (COMT)inhibitor entacapone.

The majority of these medicaments intervene in the dopaminergic and/orcholinergic signal cascade and symptomatically influence motordisturbances.

Whilst the above symptomatic treatments can enhance the life of a PDpatient, often restoring function to nearly normal for some period oftime, each have side effects and no treatment provides a cure for thedisease. Over time, as the disease progresses, drug dosing must beadjusted to best meet a patient's symptomatic needs.

SUMMARY

The present invention provides quinazoline compounds which areneurologically active and can be used in the treatment of PD.

International Patent Publication No. WO2005/095360 describesheterocyclic compounds having two fused 6-membered rings with nitrogenatoms at positions 1 and 3, a carboxy group at position 4 and a hydroxygroup at position 8 with both rings being aromatic. These compounds areuseful as pharmaceutical agents, in particular for the treatment ofneurological conditions, more specifically neurodegenerative conditionssuch as Alzheimer's disease (AD).

We have now developed heterocyclic compounds having two fused 6-memberedrings with nitrogen atoms at positions 1 and 3, a carboxy group atposition 4, a hydroxy group at position 8 and substituents at the 2 and3 positions.

These compounds fall within the generic scope of International PatentPublication No. 2005/095360, but are not specifically disclosed therein.

While not wishing to be bound by any theory, it is believed that thenature of the substituents at positions 2 and 3 may be important makingthem useful in the treatment of PD. Particularly, substituents at thesepositions are established to provide superior management of biologicallyavailable ionic iron (Fe), which has been identified in highconcentrations within the region of the human brain associated with(PD)—the substantia nigra (SN). In response to an unidentified triggerin PD, Fe levels in neurons of the SN rise significantly. This rise inFe is believed to be associated with the generation of damaging reactiveoxygen species (ROS) and may be a factor contributing to loss of thedopamine producing cells of the SN, which is the key neuropathologicalfeature of PD. The inventors have identified that the compounds of thepresent invention are specifically capable of associating principallywith Fe, acting to prevent its excessive uptake by neuronal cells andlowering its intracellular concentration.

The action of the compounds of the present invention differs from thetreatment of other neurological diseases, such as AD. Compounds employedfor AD are designed to associate principally with the ionic metalscopper (Cu) and zinc (Zn). These particular metals have beendemonstrated to be closely bound within plaque deposits of the proteinbeta amyloid (Abeta), a hallmark of AD pathology. The sequestering andremoval of Cu and Zn is considered to reduce their availability forinteraction with Abeta, inhibiting that protein's toxicity and tendencyto form plaques. Compounds for treatment of AD have further beendemonstrated to display the function of transporting Cu and/or Zn intoneuronal cells. In AD, such transported metals have been demonstrated toinfluence (decrease) the processing of the parent molecule APP, which iscleaved to produce Abeta, as seen in Alzheimer plaques and to promoteexpression of enzymes which degrade Abeta. The compounds of the presentinvention have alternatively been selected to preferentially notfacilitate cellular metal uptake.

In one aspect, there is provided a compound of general formula I

in whichR¹ and R² are independently selected from H, optionally substitutedC₁₋₆alkyl, optionally substituted C₃₋₆cycloalkyl and optionallysubstituted C₂₋₆alkynyl provided that at least one of R¹ and R² is otherthan H; orR¹ and R² together with the N atom to which they are attached form anoptionally substituted 5- or 6-membered heterocyclyl which may containat least one further heteroatom selected from N and O; andR³ is selected from optionally substituted C₁₋₆alkyl and optionallysubstituted C₃₋₆cycloalkylor pharmaceutically acceptable salts thereof,with the provisos that:

-   -   (a) when R¹ and R² are methyl, then R³ is not methyl, ethyl.        CH₂CH(CH₃)₂ or methylene cyclopropyl; and    -   (b) when R¹ is H and R² is methyl, then R³ is not methyl, propyl        or cyclopropyl,

The invention also provides a process for the preparation of thecompound of formula I defined above which comprises the steps of:

-   -   (a) cyclisation of a compound of formula III in the presence of        a source of a leaving group

in whichR³ is as defined above; andR⁸ is H or a protecting group

-   -   to prepare a compound of formula IV

in whichR³ and R⁸ are as defined above; andLG is a leaving group; and

-   -   (b) aminating the compound of formula IV with a source of NR¹R²        and deprotecting when R⁸ is a protecting group.

The invention alternatively provides a process for the preparation ofthe compound of formula I defined above which comprises the steps of:

-   -   (i) cyclisation of a compound of formula V in the presence of a        source of NR³

in which

-   -   R³ is as defined above; and    -   LG is a leaving group    -   to prepare a compound of formula VI

-   -   in which    -   R³ and LG are as defined above; and

(ii) aminating the compound of formula VI with a source of NR¹R² inwhich R¹ and R² are as defined above.

The invention further provides use of the compound of formula I definedabove as a pharmaceutical agent, preferably a neurotherapeutic orneuroprotective agent, more preferably an agent for the treatment ofParkinson's disease.

The compound of formula I is advantageously administered in the form ofa pharmaceutical composition together with a pharmaceutically acceptablecarrier.

In a second aspect, there is provided a pharmaceutical compositioncomprising the compound of formula I defined above and apharmaceutically acceptable carrier.

In a third aspect, there is provided a method for the treatment ofParkinson's disease which comprises administering an effective amount ofa compound of general formula II

in whichR¹ and R² are independently selected from H, optionally substitutedC₁₋₆alkyl and optionally substituted C₂₋₆alkynyl provided that at leastone of R¹ and R² is other than H; orR¹ and R² together with the N atom to which they are attached form anoptionally substituted 5- or 6-membered heterocyclyl which may containat least one further heteroatom selected from N and O; andR³ is selected from optionally substituted C₁₋₆alkyl and optionallysubstituted C₃₋₆cycloalkylor pharmaceutically acceptable salts thereof.

There is also provided use of the compound of formula II defined abovefor the treatment of Parkinson's disease.

There is further provided use of the compound of formula II definedabove in the manufacture of a medicament for the treatment ofParkinson's disease.

There is still further provided the compound of formula II defined abovefor use in the treatment of Parkinson's disease.

DETAILED DESCRIPTION

The present invention relates to neurologically active compounds thatare useful for the treatment of Parkinson's disease.

The present invention provides a compound of general formula I

in whichR¹ and R² are independently selected from H, optionally substitutedC₁₋₆alkyl, optionally substituted C₃₋₆cycloalkyl and optionallysubstituted C₂₋₆alkynyl provided that at least one of R¹ and R² is otherthan H; orR¹ and R² together with the N atom to which they are attached form anoptionally substituted 5- or 6-membered heterocyclyl which may containat least one further heteroatom selected from N and O; andR³ is selected from optionally substituted C₁₋₆alkyl and optionallysubstituted C₃₋₆cycloalkylor pharmaceutically acceptable salts thereof,with the provisos that:

-   -   (a) when R¹ and R² are methyl, then R³ is not methyl, ethyl,        CH₂CH(CH₃)₂ or methylene cyclopropyl and    -   (b) when R¹ is H and R² is methyl, then R³ is not methyl, propyl        or cyclopropyl.

In one embodiment, there is provided the compound of formula I definedabove, with the further proviso that:

-   -   (c) when R¹ and R² are methyl and R³ is methyl, ethyl or        methylene cyclopropyl, then pharmaceutically acceptable salts of        formula I are excluded.

In an alternative embodiment, there is provided the compound of formulaI defined above, with the further proviso that:

-   -   (c) when R¹ and R² are methyl, then R³ is not methyl, ethyl or        methylene cyclopropyl,    -   In another embodiment, there is provided the compound of formula        I defined above, with the further provisos that:    -   (d) when R¹ is H and R² is ethyl, then R³ is not ethyl; and    -   (e) when R¹ is H and R² is ethyl, then R³ is not C₁₋₂alkyl.

The optional substituents are preferably selected from C₁₋₄alkyl,optionally substituted aryl, halo, optionally substituted C₃₋₆cycloalkyland optionally substituted 5or 6-membered heterocyclyl containing atleast one heteroatom selected from N and O.

Preferably R³ is selected from methyl substituted with C₃₋₆cycloalkyl ormethyl substituted with optionally substituted 5- or 6-memberedheterocyclyl containing at least one heteroatom selected from N and O;substituted C₂₋₆alkyl and optionally substituted C₃₋₆cycloalkyl.

Preferably R¹ is H and R² is selected from C₃₋₆alkyl, substitutedC₁₋₅alkyl, optionally substituted C₁₋₅alkyl and optionally substitutedC₂₋₆alkynyl.

Preferably R¹ is C₁₋₆alkyl and R² is selected from C₂₋₆alkyl. C₁₋₆substituted alkyl and C₂₋₆alkynyl.

Preferably R¹ and R² together with the N atom to which they are attachedform an optionally substituted 5- or 6-membered heterocyclyl which maycontain at least one further heteroatom selected from N and O.

In one embodiment, the compounds of formula I have the formula Ia

in whichR¹ _(a) is selected from H and C₁₋₄alkyl;R² _(a) is selected from C₁₋₄alkyl, C₂₋₆alkynyl and C₁₋₄alkylsubstituted with optionally substituted aryl; orR¹ _(a) and R² _(a) together with the N atom to which they are attachedform an optionally substituted 5- or 6-membered heterocyclyl which maycontain at least one further heteroatom selected from N and O; andR³ _(a) is C₁₋₄alkylor pharmaceutically acceptable salts thereof,with the proviso that when R¹ _(a) and R² _(a) are methyl, then R³ _(a)is not methyl, ethyl, CH₂CH(CH₃)₂ or methylene cyclopropyl.

In another embodiment, the compounds of formula I have the formula Iadefined above, with the proviso that when R¹ _(a) is H and R² _(a) isethyl, then R³ _(a) is not C₁₋₂alkyl.

Preferably R² _(a) is selected from C₃₋₄alkyl, C₂₋₆alkynyl and C₁₋₄alkylsubstituted with optionally substituted aryl.

Preferably R¹ _(a) and R² _(a) together with the N atom to which theyare attached form an optionally substituted saturated 5- or 6-memberedheterocyclyl which may contain at least one further heteroatom selectedfrom N and O.

Representative examples of compounds of formula Ia are as follows:

or pharmaceutically acceptable salts thereof.

Preferred examples of compounds of formula Ia include F4495, F4486 andF4496, more preferably F4496.

In another embodiment, the compounds of formula I have the formula Ib

in whichR¹ and R² are as defined above; andR³ _(b) is optionally substituted C₃₋₆cycloalkylor pharmaceutically acceptable salts thereof.

Preferably R¹ and R² are independently selected from C₁₋₄alkyl.

In a further embodiment, the compounds of formula I have the formula Ic

in whichR¹ _(c) is selected from H and C₁₋₄alkyl;R² _(c) is selected from C₁₋₄alkyl. C₃₋₆cycloalkyl, methyl substitutedwith optionally substituted aryl and C₂₋₄alkynyl; orR¹ _(c) and R² _(c) together with the N atom to which they are attachedform an optionally substituted 5- or 6-membered heterocyclyl which maycontain at least one further heteroatom selected from N and O; andR³ _(c) is selected from optionally substituted C₃₋₈cycloalkyl,optionally substituted aryl and optionally substituted 5- or 6-memberedheterocyclyl containing at least one heteroatom selected from N and O orpharmaceutically acceptable salts thereof.

Preferably aryl in R² _(c) and R³ _(c) is optionally substituted withhalo.

Preferably R¹ _(c) is H and R² _(c) is C₃₋₄alkyl.

Preferably R¹ _(c) is C₁₋₆alkyl and R² _(c) is selected from C₁₋₆alkyland C₂₋₆alkynyl.

Representative examples of compounds of formula Ic are as follows:

or pharmaceutically acceptable salts thereof.

The present invention also provides a method for the treatment ofParkinson's disease which comprises administering an effective amount ofa compound of general formula II

in whichR¹ and R² are independently selected from H, optionally substitutedC₁₋₆alkyl and optionally substituted C₂₋₆alkynyl provided that at leastone of R¹ and R² is other than H; orR¹ and R² together with the N atom to which they are attached form anoptionally substituted 5- or 6-membered heterocyclyl which may containat least one further heteroatom selected from N and O; andR³ is selected from optionally substituted C₁₋₆alkyl and optionallysubstituted C₃₋₆cycloalkylor pharmaceutically acceptable salts thereof.

Representative examples of compounds of formula II in addition to thoseof formulae Ia and Ic above include the following

or pharmaceutically acceptable salts thereof.

The term “C₁₋₆alkyl” refers to straight chain or branched chainhydrocarbon groups having from 1 to 6 carbon atoms. Examples includeethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, neopentyl and hexyl.

The term “C₁₋₆alkylene” refers to the divalent equivalent of “C₁₋₆alkyl”defined above.

The term “C₂₋₆alkynyl” refers +to straight chain or branched chainhydrocarbon groups having at least one triple bond and 2 to 4 carbonatoms. Examples include ethynyl, 1- or 2-propynyl, 1-, 2- or 3-butynyland methyl-2-propynyl.

The term “C₃₋₆cycloalkyl” refers to non-aromatic cyclic hydrocarbongroups having from 3 to 6 carbon atoms. Examples include cyclopropyl,cyclobutyl, cyclopentyl and cyclohexyl.

The term “aryl” refers to single, polynuclear, conjugated or fusedresidues of aromatic hydrocarbons. Examples include phenyl, biphenyl,terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl,dihydroanthracenyl, benzanthracenyl, dibenxanthracenyl andphenanthrenyl. 6-membered aryls such as phenyl are preferred.

The term “5- or 6-membered heterocyclyl” refers to saturated orunsaturated monocyclic hydrocarbon groups containing at least oneheteroatom atom selected from the group consisting of N and O.

Suitable heterocyclyls include N-containing heterocyclic groups, suchas, unsaturated 5- or 6-membered heteromonocyclic groups containing 1 to4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl ortetrazolyl;

saturated 5 or 6-membered heteromonocyclic groups containing 1 to 3nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidinyl orpiperazinyl;

unsaturated 5- to 6-membered heteromonocyclic group containing an oxygenatom, such as, pyranyl or furyl;

unsaturated 5 or 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl oroxadiazolyl; and

saturated 5- or 6-membered heteromonocyclic group containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl.

Preferred heterocyclyls include piperidinyl, piperazinyl, morpholinyl,pyridinyl, pyrazolyl, imidazolyl and tetrazolyl.

The term “halo” refers to fluorine, chlorine, bromine and iodine,preferably fluorine.

The term “optionally substituted” refers to a group that may or may notbe further substituted with one or more groups selected from C₁₋₆ alkyl,Si(C₁₋₆alkyl)₃, C₃₋₆ cycloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, aryl,heterocycylyl, halo, haloC₁₋₆alkyl, haloC₃₋₆cycloalkyl, haloC₂₋₆alkenyl,haloC₂₋₆alkynyl, haloaryl, haloheterocycylyl, hydroxy, C₁₋₆ alkoxy,C₂₋₆alkenyloxy, C₂₋₆alkynyloxy, aryloxy, heterocyclyloxy, carboxy,haloC₁₋₆alkoxy, haloC₂₋₆alkenyloxy, haloC₂₋₆alkynyloxy, haloaryloxy,nitro, nitroC₁₋₆alkyl, nitroC₂₋₆alkenyl, nitroaryl, nitroheterocyclyl,azido, amino, C₁₋₆alkylamino, C₂₋₆alkenylamino, C₂₋₆alkynylamino,arylamino, heterocyclamino acyl, C₁₋₆alkylacyl, C₂₋₆alkenylacyl,C₂₋₆alkynylacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy,aldehydro, C₁₋₆alkylsulfonyl, arylsulfonyl, C₁₋₆alkylsulfonylamino,arylsulphonylamino, C₁₋₆alkylsulfonyloxy, arylsulfonyloxy,C₁₋₆alkylsulfenyl, C₂₋₆alklysulfenyl, arylsulfonyl, carboalkoxy,carboaryloxy, mercapto, C₁₋₆alkylthio, arylthio, acylthio, cyano,phosphorus-containing groups and the like. Preferred optionalsubstituents are selected from C₁₋₄alkyl, optionally substituted aryl,halo, optionally substituted C₃₋₆cycloalkyl and optionally substituted5- or 6-membered heterocyclyl containing at least one heteroatomselected from N and O.

The compounds of the invention may also be prepared as salts which arepharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts. Examples ofpharmaceutically acceptable salts include salts of pharmaceuticallyacceptable cations such as sodium, potassium, lithium, calcium,magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamicand hydrobromic acids; or salts of pharmaceutically acceptable organicacids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulfonic, trihalomethanesulfonic,toluenesulfonic, benzenesulfonic, isethionic, salicylic, sulphanilic,aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric,pantothenic, tannic, ascorbic, valeric and orotic acids. Salts of aminegroups may also comprise quaternary ammonium salts in which the aminonitrogen atom carries a suitable organic group such as an alkyl,alkenyl, alkynyl or aralkyl moiety. Preferred salts are acid additionsalts of pharmaceutically acceptable inorganic acids such ashydrochloric or hydrobromic acid addition salts.

The salts may be formed by conventional means, such as by reacting thefree base form of the compound with one or more equivalents of theappropriate acid in a solvent or medium in which the salt is insoluble,or in a solvent such as water which is removed in vacuo or by freezedrying or by exchanging the anions of an existing salt for another anionon a suitable ion exchange resin.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms or crystal formsthereof, particularly solvates or polymorphs. Solvates contain eitherstoichiometric or non-stoichiometric amounts of a solvent, and may beformed during the process of crystallization with pharmaceuticallyacceptable solvents such as water, alcohols such as methanol, ethanol orisopropyl alcohol, DMSO, acetonitrile, dimethyl formamide (DMF) and thelike with the solvate forming part of the crystal lattice by eithernon-covalent binding or by occupying a hole in the crystal lattice.Hydrates are formed when the solvent is water, or alcoholates are formedwhen the solvent is alcohol. Solvates of the compounds of the presentinvention can be conveniently prepared or formed during the processesdescribed herein. In addition, the compounds of the present inventioncan exist in unsolvated as well as solvated forms. In general, thesolvated forms are considered equivalent to the unsolvated forms for thepurposes of the compounds and methods provided herein.

Additionally, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. The solvated forms of thecompounds of the present invention are also considered to be disclosedherein.

Where a compound possesses a chiral center the compound can be used as apurified enantiomer or diastereomer, or as a mixture of any ratio ofstereoisomers. It is however preferred that the mixture comprises atleast 70%, 80%, 90%, 95%, 97.5% or 99% of the preferred isomer.

This invention also encompasses prodrugs of the compounds of formula I.

The term “pro-drug” is used herein in its broadest sense to includethose compounds which are converted in vivo to the compound of formulaI. Use of the pro-drug strategy optimises the delivery of the drug toits site of action, for example, the brain. In one embodiment, compoundsof formula I having free amino, amido, hydroxy or carboxylic acid groupscan be converted into prodrugs. Prodrugs include compounds whereincarbonates, carbamates, amides and alkyl esters which are covalentlybonded to the above substituents of compounds of the present inventionthrough the carbonyl carbon prodrug sidechain. Prodrugs also includephosphate derivatives of compounds (such as acids, salts of acids, oresters) joined through a phosphorus-oxygen bond to a free hydroxyl ofcompounds of formula I. Prodrugs may also include N-oxides, and S-oxidesof appropriate nitrogen atoms in formula I.

In another embodiment, prodrugs include the presence of a C₁₋₆ alkyl orarylester moiety which is designed to resist hydrolysis until thepro-drug has crossed the BBB, where esterases on the inner surface ofthe BBB act to hydrolyse the ester and liberate the C8 hydroxyl of thecompound of formula I.

Process of Making Compounds

Compounds of the general formula I are generally prepared by cyclisingthe compound of formula III. The cyclisation may be performed using anysuitable known technique for example, dehydrative cyclisation involvingchloroacetylchloride.

The products formed from either reaction step may be further derivatisedusing techniques known to those skilled in the art. Alternatively,derivatisation of the mono-halo intermediate may be undertaken prior toreaction of the second halo substituent. Those skilled in the art willappreciate that the order of the reactions described for the synthesesabove may be changed in certain circumstances and that certainfunctionalities may need to be derivatised (i.e. protected) in certaininstances for the reactions described above to proceed with reasonableyield and efficiency. The types of protecting functionalities are wellknown to those skilled in the art and are described for example inGreene (Greene, T., Wuts, P. (1999) Protective Groups in OrganicSynthesis. Wiley-Interscience; 3rd edition).

Examples of protecting groups which may be used to protect a hydroxygroup include, but are not limited to, silyl groups (eg trimethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl), benzyl groups (eg benzyl,methoxybenzyl, nitrobenzyl, benzyl halides such as benzyl bromide orbenzyl chloride), alkyl groups (eg methyl, ethyl, n- and i-propyl, andn-, sec- and t-butyl) and acyl groups (e.g. acetyl such as acetylchloride or acetyl anhydride and benzoyl).

The leaving group may be any suitable known type such as those disclosedin J. March, “Advanced Organic Chemistry: Reactions, Mechanisms andStructure” 4^(th) Edition, pp 352-357, John Wiley & Sons, New York, 1992which is incorporated herein by reference. Preferably, the leaving groupis halogen, more preferably chlorine.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising atleast one of the compounds of the formula I and a pharmaceuticallyacceptable carrier. The carrier must be “pharmaceutically acceptable”means that it is compatible with the other ingredients of thecomposition and is not deleterious to a subject. The compositions of thepresent invention may contain other therapeutic agents as describedbelow, and may be formulated, for example, by employing conventionalsolid or liquid vehicles or diluents, as well as pharmaceuticaladditives of a type appropriate to the mode of desired administration(for example, excipients, binders, preservatives, stabilizers, flavours,etc.) according to techniques such as those well known in the art ofpharmaceutical formulation (See, for example, Remington: The Science andPractice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

The compounds of the invention may be administered by any suitablemeans, for example, orally, such as in the form of tablets, capsules,granules or powders; sublingually; buccally; parenterally, such as bysubcutaneous, intravenous, intramuscular, intra(trans)dermal, orintracisternal injection or infusion techniques (e.g., as sterileinjectable aqueous or non-aqueous solutions or suspensions); nasallysuch as by inhalation spray or insufflation; topically, such as in theform of a cream or ointment ocularly in the form of a solution orsuspension; vaginally in the form of pessaries, tampons or creams; orrectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. The compounds may, for example, be administered in a formsuitable for immediate release or extended release. Immediate release orextended release may be achieved by the use of suitable pharmaceuticalcompositions comprising the present compounds, or, particularly in thecase of extended release, by the use of devices such as subcutaneousimplants or osmotic pumps.

The pharmaceutical compositions for the administration of the compoundsof the invention may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.These methods generally include the step of bringing the compound offormula I into association with the carrier which constitutes one ormore accessory ingredients. In general, the pharmaceutical compositionsare prepared by uniformly and intimately bringing the compound offormula I into association with a liquid carrier or a finely dividedsolid carrier or both, and then, if necessary, shaping the product intothe desired formulation. In the pharmaceutical composition the activeobject compound is included in an amount sufficient to produce thedesired effect upon the process or condition of diseases. As usedherein, the term “composition” is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts.

The pharmaceutical compositions containing the compound of formula I maybe in a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents such as sweetening agents,flavouring agents, colouring agents and preserving agents, e.g. toprovide pharmaceutically stable and palatable preparations. Tabletscontain the compound of formula I in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the compound of formula I is mixed with an inert solid diluent,for example, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the compound of formula I is mixed with wateror an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compound of formulaI in a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the compound of formula I inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectable formulations.

For administration to the respiratory tract, including intranasaladministration, the active compound may be administered by any of themethods and formulations employed in the art for administration to therespiratory tract.

Thus in general the active compound may be administered in the form of asolution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for exampleprepared from water alone (for example sterile or pyrogen-free water) orwater and a physiologically acceptable co-solvent (for example ethanol,propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipientsfor example preservatives (such as benzalkonium chloride), solubilisingagents/surfactants such as polysorbates (eg. Tween 80, Span 80,benzalkonium chloride), buffering agents, isotonicity-adjusting agents(for example sodium chloride), absorption enhancers and viscosityenhancers. Suspensions may additionally contain suspending agents (forexample microcrystalline cellulose and carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase a means of dose metering is desirably provided. In the case of adropper or pipette this may be achieved by the subject administering anappropriate, predetermined volume of the solution or suspension. In thecase of a spray this may be achieved for example by means of a meteringatomising spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the compound is provided in apressurised pack with a suitable propellant, such as achlorofluorocarbon (CFC), for example dichlorodifluoromethane,trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of active compound may becontrolled by provision of a metered valve.

Alternatively the active compound may be provided in the form of a drypowder, for example a powder mix of the compound in a suitable powderbase such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP).Conveniently the powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form, for example incapsules or cartridges of eg. gelatin, or blister packs from which thepowder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the active compound will generallyhave a small particle size, for example of the order of 5 microns orless. Such a particle size may be obtained by means known in the art,for example by micronisation.

When desired, formulations adapted to give sustained release of theactive compound may be employed.

The active compound may be administered by oral inhalation as afree-flow powder via a “Diskhaler” (trade mark of Glaxo Group Ltd) or ameter dose aerosol inhaler.

The compounds of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of the present invention are employed.(For purposes of this application, topical application shall includemouthwashes and gargles.)

For application to the eye, the active compound may be in the form of asolution or suspension in a suitable sterile aqueous or non-aqueousvehicle. Additives, for instance buffers, preservatives includingbactericidal and fungicidal agents, such as phenyl mercuric acetate ornitrate, benzalkonium chloride, or chlorohexidine and thickening agentssuch as hypromellose may also be included.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolisable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilisers, preservatives,excipients and the like. The preferred lipids are the phospholipids andphosphatidyl cholines, both natural and synthetic. Methods to formliposomes are known in the art.

Efficacy of this class of compounds may be applicable to drug elutingstents. Potential applications of drug eluting stents with thesecompounds include pulmonary artery stenosis, pulmonary vein stenosis, aswell as coronary artery stenosis. Drug eluting stents may also be usedin saphenous vein grafts or arterial grafts or conduits. Drug elutingstents that release this class of compounds may also be applicable fortreating stenoses of the aorta or peripheral arteries, such as the iliacartery, the femoral artery or the popliteal artery. The compound may bebound to the drug eluting stent by any of various methods known in thefield. Examples of such methods include polymers, phosphoryl choline,and ceramics. The compound may also be impregnated into a bioabsorbablestent.

The active compounds may also be presented for use in the form ofveterinary compositions, which may be prepared, for example, by methodsthat are conventional in the art. Examples of such veterinarycompositions include those adapted for:

(a) oral administration, external application, for example drenches(e.g. aqueous or non-aqueous solutions or suspensions); tablets orboluses; powders, granules or pellets for admixture with feed stuffs;pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscularor intravenous injection, e.g. as a sterile solution or suspension; or(when appropriate) by intramammary injection where a suspension orsolution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied tothe skin; or

(d) rectally or intravaginally, e.g. as a pessary, cream or foam.

The pharmaceutical composition and method of the present invention mayfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of neurological conditions.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

Examples of other therapeutic agents include dopamine-replacing agentsand dopamine agonists such asrotigotine, pramipexole, bromocriptine,ropinirole, cabergoline, pergolide, apomorphine and lisuride,anticholinergic agents, NMDA antagonists, beta-blockers as well as theMAO-B inhibitor selegeline and the COMT inhibitor entacapone.

When other therapeutic agents are employed in combination with thecompounds of the present invention they may be used for example inamounts as noted in the Physician Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art.

Methods of Treatment

The compounds of formula I may be used in the treatment of Parkinson'sdisease.

Generally, the term “treatment” means affecting a subject, tissue orcell to obtain a desired pharmacological and/or physiological effect andinclude: (a) preventing the disease from occurring in a subject that maybe predisposed to the disease, but has not yet been diagnosed as havingit; (b) inhibiting the disease, i.e., arresting its development; or (c)relieving or ameliorating the effects of the disease, i.e., causeregression of the effects of the disease.

The term “subject” refers to any animal having a disease which requirestreatment with the compound of formula I.

In addition to primates, such as humans, a variety of other mammals canbe treated using the compounds, compositions and methods of the presentinvention. For instance, mammals including, but not limited to, cows,sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine,ovine, equine, canine, feline, rodent or murine species can be treated.However, the invention can also be practiced in other species, such asavian species (e.g., chickens).

The term “administering” should be understood to mean providing acompound of the invention to a subject in need of treatment.

Dosages

The term “therapeutically effective amount” refers to the amount of thecompound of formula I that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought bythe researcher, veterinarian, medical doctor or other clinician.

In the treatment or prevention of conditions which require kinaseinhibition an appropriate dosage level will generally be about 0.01 to500 mg per kg patient body weight per day which can be administered insingle or multiple doses. Preferably, the dosage level will be about 0.1to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kgper day. A suitable dosage level may be about 0.01 to 250 mg/kg per day,about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day.Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50mg/kg per day. For oral administration, the compositions are preferablyprovided in the form of tablets containing 1.0 to 1000 milligrams of theactive ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0,800.0, 900.0, and 1000.0 milligrams of the active ingredient. The dosagemay be selected, for example to any dose within any of these ranges, fortherapeutic efficacy and/or symptomatic adjustment of the dosage to thepatient to be treated. The compounds will preferably be administered ona regimen of 1 to 4 times per day, preferably once or twice per day.

It will be understood that the specific dose level and frequency ofdosage for any particular patient may be varied and will depend upon avariety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

EXAMPLES

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

For clarity, compounds of this invention are referred to by number, forexample 1-4 and 2-3. The structures of the example compounds so referredto are given in Tables 1-7.

In Examples 1 to 7, the following reference is cited:

-   White et al., J Neuroscience, (1998) 18, 6207-6217.

Example 1 Scheme 1

A range of 2,3-disubstituted 8-hydroxy-3H-quinazolin-4-ones 4-9 can beprepared by the synthetic route depicted in Scheme 1. Commerciallyavailable 2,4-dichlorobenzoic acid 1-1 is nitrated to give2,4-dichloro-5-nitrobenzoic acid 1-2. Nitro compound 1-2 is reduced withtin(II)chloride to provide aniline 1-3 which in turn is converted to theacetamide 1-4. A second nitration provides intermediate 1-5, subsequentbase hydrolysis affords phenol 1-6. Amide 2-10 is produced in thepresence of an amine and activating agent, EDC. The reaction isnoteworthy in that the phenol does not require protection. Amide 2-10 isallowed to react with Fe powder in glacial acetic acid to give amine2-11. Reaction with chloroacetyl chloride followed by condensationreaction with PCl₃ affords chloromethyl compound 4-15. Finally,amination with a secondary amine and HCl formation provides the desiredtarget compounds 4-9.

2,4-Dichloro-5-nitrobenzoic Acid (1-2)

2,4-Dichlorobenzoic acid 1-1 (1.0 g, 5.34 mmol) was added in one portionto a stirred solution of concentrated nitric acid (0.26 mL, 5.75 mmol)in concentrated sulphuric acid (7 mL). After 3 hours at room temperaturethe solution was poured onto ice. The resulting white solid was isolatedvia filtration and washed with H₂O (×3). The white solid was thenstirred with 1% Na₂CO₃ (aq) (20 mL) for 1 h at room temperature.Remaining insoluble material was filtered off and the resulting clearfiltrate was concentrated to a pale yellow solid. Recrystallisation fromH₂O afforded the sodium salt as pale yellow crystals. (Note: the otherminor product, sodium 2,4-dichloro-3-nitrobenzoate, remained insolution). The yellow crystals of sodium 2,4-dichloro-5-nitrobenzoatewas dissolved in a minimum amount of H₂O and acidified by the slowaddition of concentrated HCl until a white precipitate formed. Afterisolation by filtration and washing with H₂O 2,4-dichloro-5-nitrobenzoicacid 1-2 was obtained as a white solid (0.91 g, 72%). ¹H NMR (400 MHz,CDCl₃) δ7.73 (s, 1H), 8.60 (s, 1H).

2,4-Dichloro-5-aminobenzoic Acid (1-3)

Tin(II)Chloride hydrate (50 g, 0.29 mol) was added to a solution of2,4-dichloro-5-nitrobenzoic acid 1-2 (10.0 g, 0.045 mol) in EtOH (200mL). The mixture was stirred for 70° C. for 30 min, cooled and pouredonto ice. The pH of the mixture was adjusted to 8 using saturatedaqueous NaHCO₃. The suspension was left to stir at room temperature for5 h and re-acidified to pH 5 with glacial AcOH. The resulting whitesuspension was continuously washed with EtOAc, the extracts combined,washed with brine, dried over Na₂SO₄, filtered and concentrated toafford the desired amine 1-3 as an off-white solid (8.8 g, 96%). ¹H NMR(400 MHz, CD₃OD) δ7.27 (s, 1H), 7.30 (s, 1H).

5-Acetamido-2,4-dichlorobenzoic Acid (1-4)

Acetic anhydride (27 mL) was added to 2,4-dichloro-5-aminobenzoic Acid1-3 (8.0 g, 0.041 mol) in glacial AcOH (150 mL). The solution wasstirred at room temperature for 30 min and concentrated to yield thedesired acetamide 1-4 as a white solid (9.6 g, 96%). ¹H NMR (400 MHz,CD₃OD) δ2.19 (s, 3H), 7.62 (s, 1H), 8.32 (s, 1H).

3-Acetamido-4,6-dichloro-2-nitrobenzoic Acid (1-5)

5-Acetamido-2,4-dichlorobenzoic acid 1-4 (40.0 g, 0.161 mol) was addedin portions to a cooled mixture of concentrated H₂SO₄ (400 mL) andconcentrated nitric acid (400 mL) at 5° C. After stirring for 2 hconcentrated H₂SO₄ (100 mL)/concentrated nitric acid (100 mL) was added.After another 90 min concentrated H₂SO₄ (400 mL)/concentrated nitricacid (200 mL) was added and stirring was continued for a further 2 h.The reaction mixture was cautiously poured onto ice resulting in theformation of a yellow precipitate. The product was collected byfiltration then extracted into EtOAc. The combined organic layers werewashed with H₂O, brine, dried over Na₂SO₄, filtered and concentrated toprovide the nitro compound 1-5 as a yellow/orange solid (38.1 g, 81%yield). ¹H NMR (400 MHz, d6-DMSO) δ1.99 (s, 3H), 8.21 (s, 1H), 10.27 (s,1H).

4,6-Dichloro-3-hydroxy-2-nitrobenzoic Acid (1-6)

A solution of KOH (73.4 g) in H₂O (330 mL) was added to3-acetamido-4,6-dichloro-2-nitrobenzoic acid 1-5 (38.1 g, 0.130 mol).The resulting brown solution was heated to reflux for 18 h (bath temp130° C.). The reaction was cooled and extracted with ether. The aqueouslayer was then acidified to pH 2 with concentrated HCl and extractedinto EtOAc (×4). The combined organic layers were washed with brine(×3), dried over Na₂SO₄, filtered and concentrated to provide4,6-dichloro-3-hydroxy-2-nitrobenzoic acid 1-6 as a brown solid (31.0 g,95%). ¹H NMR (400 MHz, d6-DMSO) δ7.88 (s, 1H).

4,6-Dichloro-N-cyclopropylmethyl-3-hydroxy-2-nitrobenzamide (2-10,R=cyclopropylmethyl))

The 4,6-dichloro-3-hydroxy-2-nitrobenzoic acid 1-6 (5.03 g, 19.9 mmol)was dissolved in anhydrous CH₂Cl₂ (60 mL) and anhydrous THF (30 mL) thentreated with EDC (4.61 g, 24.0 mmol), (aminomethyl)cyclopropane (2.35mL, 27.1 mmol) and DIEA (4.18 mL, 24.0 mmol). The reaction was stirredovernight at room temperature then diluted with CH₂Cl₂ and 0.5M HCl(aq). The product was extracted into CH₂Cl₂ (×4). The organic layer waswashed with sat.aq NaHCO₃, brine, dried over Na₂SO₄, filtered andconcentrated to afford the amide 2-10 as a brown foam (3.40 g, 56% crudeyield). ¹H NMR (400 MHz, d6-DMSO) δ0.17 (m, 2H). 0.32 (m, 2H), 0.93 (m,1H), 3.0 (t, J=8 Hz, 2H), 7.16 (s, 1H), 8.38 (br t, J=8.0 Hz, 1H).

2-Amino-4,6-dichloro-N-cyclopropylmethyl-3-hydroxy-benzamide (2-11,R=cyclopropylmethyl)

The amide 2-10 (10.1 g, 33.1 mmol) was dissolved in glacial AcOH (180mL) then treated with Fe powder (11.2 g, 0.2 mol) and heated to 80° C.under nitrogen for 1.5 h. The beige suspension was treated with sat. aqNaHCO₃ until clear then filtered through celite washing with EtOH. Thefiltrate was concentrated then extracted into EtOAc (×4). The organiclayer was washed with brine, dried over Na₂SO₄, filtered, concentratedand triturated with ether/petrol (1:10). The amine 2-11 was collected byfiltration as dark brown solid (6.8 g, 75%). ¹H NMR (400 MHz, d6-DMSO)δ0.21 (m, 2H), 0.39 (m, 2H), 1.18 (m, 1H), 3.10 (t, J=8.0 Hz, 2H), 4.93(br s, 2H), 6.84 (s, 1H), 8.42 (br t, J=8.0 Hz, 1H).

5,7-Dichloro-2-chloromethyl-3-cyclopropylmethyl-8-hydroxy-3H-quinazolin-4-one(4-15, R=cyclopropylmethyl)

The amine 2-11 (6.8 g, 24.7 mmol) in anhydrous CH₂Cl₂ (85 mL) wastreated with chloroacetyl chloride (4.5 mL, 56.5 mmol) at 0° C. underN₂. The reaction was warmed to room temperature for 30 min then dilutedwith ether (30 mL)/petrol (100 mL). The resulting brown precipitate wascollected by filtration, washing with petrol and dried to provide thechloromethylacetamide intermediate as a brown powder (6.4 g, 74%). ¹HNMR (400 MHz, d6-DMSO) δ 0.18 (m, 2H), 0.38 (m, 2H), 1.26 (m, 1H), 3.04(t, J=8.0 Hz, 2H), 4.19 (s, 2H), 7.52 (s, 1H), 8.18 (s, 1H), 9.67 (s,1H), 10.07 (br s, 1H). To a suspension of the chloromethylacetamide(2.59 g, 7.37 mmol) in toluene (73 mL) was added PCl₃ (1.93 mL, 22.1mmol). The reaction was heated to vigorous reflux under Ar for 2.5 h.Cooled and concentrated in vacuo. Added H₂O (10 mL) then sat. aq NaHCO₃(2 mL) until ph 5-6. Added EtOAc and sonicated well, transferring themixture into a separating funnel. Extracted into EtOAc (×4), dried overNa₂SO₄, filtered, concentrated and purified by FC eluting with 20%EtOAc/petrol to provide the desired chloromethyl compound 4-15 as alight orange solid (771 mg, 31%). ¹H NMR (400 MHz, d6-DMSO) δ0.41 (m,4H), 1.22 (m, 1H), 3.97 (d, J=4.4 Hz, 2H), 4.81 (s, 2H), 7.57 (s, 1H),10.39 (s, 1H).

F4267

Chloromethyl derivative 4-15 (917 mg, 2.75 mmol) was dissolved inanhydrous CH₂Cl₂ (25 mL) cooled to 0° C. then treated with dimethylamine(7.5 mL, 15.0 mmol, 2.0M solution in MeOH). The reaction was allowed towarm to room temperature overnight then concentrated and extracted intoCH₂Cl₂ washing with sat. aq. NaHCO₃. The aqueous layer was furtherextracted with CH₂Cl₂ (×3). The combined organic layers were dried overNa₂SO₄, filtered and concentrated. The resulting oil was taken up inMeOH (50 mL) and treated with conc. HCl (1.5 mL). After concentratingthe solution the resulting yellow solid was pumped dry and thentriturated with MeOH (3 mL)/ether (70 mL) to precipitate F4267 (820 mg,79%) as pale yellow solid that was collected by filtration (See Table 1for Analysis).

TABLE 1 Compounds prepared according to Example 1 (Scheme 1) CompoundStructure MW ¹H NMR Mass Spec F4267

Parent: 342.2  HCl salt: 378.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.48 (m,4H), 1.19 (t, J = 4.80 Hz, 1H), 2.94 (s, 3H), 2.95 (s, 3H), 3.85 (d, J =6.8 Hz, 2H), 4.86 (d, J = 4.80 Hz, 2H), 7.63 (s, 1H), 8.68 (br s , 1H)m/z 342.1, 344.1 [M + H]⁺ F4268

Parent: 316.2  HCl Salt 352.6  ¹H NMR (400 MHz, d6-DMSO) δ 1.24 (t, J =7.2 Hz, 3H), 2.96 (s, 3H), 2.97 (s, 3H), 3.91 (q, J = 7.2 Hz, 2H), 4.79(d, J = 5.2 Hz, 2H 7.64 (s, 1H), 10.21 (s, 1H), 10.51 (s, 1H). m/z316.1, 318.0 [M + H]⁺ F4383

Parent: 411.3  bis HCl Salt: 484.3  ¹H NMR (400 MHz, D₂O) δ 0.20 (d, J =4.0 Hz, 2H), 0.39 (d, J = 4.8 Hz, 2H), 0.96 (m, 1H), 1.17 (t, J = 7.0Hz, 3H), 3.03 (q, J = 7.0, 4.0 Hz, 2H), 3.30 (br m, 8H), 3.78 (d, J =4.0 Hz, 2H), 4.06 (s, 2H), 7.04 (s, 1H). m/z 411.2, 413.2 [M + H]⁺ F4384

Parent: 384.3  HCl salt 420.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.48 (m, 4H),1.21 (m, 1H), 3.59 (d, J = 4.8 Hz, 2H), 3.90 (d, J = 4.0 Hz, 2H), 3.97(d, J = 4.8 Hz, 2H), 4.15 (t, J = 4.8 Hz, 2H), 4.85 (s, 2H), 7.62 (s,1H), 10.58 (s, 1H), 10.87 (br s, 1H). m/z 411.2, 413.2 [M + H]⁺ F4385

Parent: 370.3  HCl salt: 406.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.43 (m,2H), 1.17 (t, J = 7.6 Hz, 2H), 1.20 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H),2.86 (m, 2H), 3.15 (partially obscured), 3.92 (d, J = 7.2 Hz, 2H), 4.76(d, J = 4.8 Hz, 2H), 7.65 (s, 1H), 10.10 (s, 1H), 10.81 (s, 1H). m/z370.1, 372.2 [M + H]⁺ F4386

Parent: 344.2  HCl salt: 380.7  ¹H NMR (400 MHz, d6-DMSO) δ 1.25, (t, J= 7.2 Hz), 1.30 (t, J = 7.2 Hz, 3H), 4.71 (d, J = 4.0 Hz, 2H), 3.38(partially obscured), 3.96 (q, J = 7.2, 4.0 Hz, 2H), 7.65 (s, 1H), 9.66(s, 1H), 10.63 (s, 1H). m/z 344.0, 346.1 [M + H]⁺ F4387

Parent: 366.2  HCl salt: 402.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.46 (d, J =4.4 Hz, 2H), 0.52 (d, J = 7.2 Hz, 2H), 1.18 (m, 1H), 3.03 (br s, 3H),3.90 (m, 2H), 3.98 (s, 1H), 4.32 (s, 2H), 4.84 (s, 2H), 7.66 (s, 1H),10.51 (s, 1H), 10.68 (s, 1H). m/z 366.1, 368.1 [M + H]⁺ F4391

Parent: 382.3  HCl salt 418.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.53 (m, 4H),1.19 (m, 1H), 1.43 (m, 1H), 1.72 (m, 1H), 1.81 (m, 2H), 2.06 (m, 2H),3.18 (m, 2H), 3.67 (m, 2H), 3.92 (d, J = 7.0 Hz), 4.80 (s, 2H), 7.61 (s,1H), 10.18 (br s, 1H), 10.65 (s, 1H). m/z 382.4, 384.4 F4392

Parent: 340.2  HCl salt: 376.7  ¹H NMR (400 MHz, d6-DMSO) δ 1.24 (t, J =7.2 Hz, 3H), 3.03 (s, 3H), 3.96 (m, 3H), 4.30 (s, 2H), 4.80 (s, 2H),7.65 (s, 1H), 10.50 (s, 1H), 10.65 (br s, 1H). m/z 340.1, 342.1 [M + H]⁺F4480

Parent: 436.31 HCl salt: 472.8  ¹H NMR (400 MHz, d6-DMSO) δ 0.37 (d, J =4.4 Hz, 2H), 0.46 (d, J = 7.6 Hz, 2H), 1.07 (m, 1H), 2.96 (d, J = 4.4Hz, 3H), 3.79 (m, 2H), 4.48 (app t, J = 4.4 Hz, 2H), 4.73 (s, 2H), 7.20(app t, J = 8.4 Hz, 2H), 7.60 (s, 1H), 7.64 (app t, J = 7.2 Hz, 2H),10.23 (br s, 1H), 10.51 (s, 1H). m/z 436.4, 438.4 [M + H]⁺ F4499

Parent: 368.3  HCl salt 404.7  ¹H NMR (400 MHz, d6-DMSO) δ 0.45 (d, J =3.6 Hz, 2H), 0.48 (d, J = 7.2 Hz, 2H), 1.21 (m, 1H), 2.05 (br m, 4H),3.26 (m, 2H), 3.74 (m, 2H), 3.86 (d, J = 8.0 Hz, 2H), 4.90 (d, J = 4.2Hz, 2H), 7.65 (s, 1H), 10.51 (s, 1H), 10.58 (br s, 1H). m/z 368.4, 370.4[M + H]⁺ F4535

Parent: 398.33 HBr Salt: 434.79 ¹H NMR (400 MHz, d6-DMSO) δ 0.41-0.46(m, 4H), 1.19 (m, 1H), 1.73 (m, 4H), 3.19 (m, 4H), 3.86 (d, J = 7.2 Hz,2H), 4:74 (s, 2H), 7.60 (s, 1H), 10.18 (bs, 1H), 10.77 (s, 1H) m/z 400.2[M + H]⁺, 402.2 F4536

Parent 410.34 HCl Salt: 446.60 ¹H NMR (d6-DMSO, 400 MHz) □ 0.50 (m, 4H),1.08 (t, J = 6.8 Hz, 2H), 1.21 (m, 5H), 1.58 (m, 2H), 1.79 (m, 2H), 2.17(m, 2H), 2.93 (s, 3H), 3.92 (s, 2H), 4.48 (m, 1H), 4.87 (d, J = 16.8 Hz,1H), 7.66 (s, 1H), 9.82 br m, 1H), 10.78 (d, J = 11.6 Hz, 1H). ES + ve410.5, 412.4 [M + H]⁺ F4581

Parent: 356.25 HBr Salt: 392.71 ¹H NMR (500 MHz, d6-DMSO) δ 0.46-0.53(m, 4H), 1.23 (m, 1H), 1.37: (t, J = 7.0 Hz, 3H), 2.95 (d, 4J = 5.0 Hz,3H), 3.33 (m, 1H), 3.41 (m, 1H), 3.91 (d, J = 7.0 Hz, 2H), 4.74 (dd, 2J= 17.0, 4J = 6.0 Hz, 1H), 4.88 (dd, 2J = 17.0, 4J = 4.0 Hz, 1H), 7.65(s, 1H), 10.39 (bs, 1H), 10.70 (s, 1H). m/z 356.3, 358.3 [M + H]⁺ F4H

Parent: 328.19 F4I

Parent: 342.22 F4J

Parent: 356.25

Example 2 Scheme 2

A range of novel 2,3-disubstituted 8-hydroxy-3H-quinazolin-4-ones 4-9can be prepared by the synthetic route depicted in Scheme 2. Nitro acid1-6 prepared according to Scheme 1 shown in Example 1 is selectivelymethylated at the carboxy group using iodomethane in DMF to affordmethyl ester 1-7. Conversion to the methyl ether 2-2 is achieved byheating 1-7 in acetone and iodomethane. Hydrolysis of 2-2 by heating inNaOH and methanol affords the acid 2-1. Acid chloride formation followedby reaction with an amine provides intermediate 4-5 in good to excellentyields. Reduction with Fe powder in glacial acetic acid gives the amine4-6 which in turn is cyclised to 4-7 in refluxing AcOH andchloroacetylchloride. Amination of 4-7 followed by deprotection witheither BBr₃ in CH₂Cl₂ or refluxing HBr affords target compound 4-9.

4,6-Dichloro-3-methoxy-2-nitrobenzoic Acid (1-7)

To a solution of acid 1-6 (0.88 g, 3.5 mmol) in DMF (8 mL) was addedK₂CO₃ (1.44 g, 10 mmol) followed by iodomethane (0.43 mL, 6.95 mmol).The reaction was heated to 60° C. for 17 h then DMF was removed underreduced pressure to give an orange gum. H₂O (20 mL) was added and thesolution was acidified to pH 1 with a 10% HCl solution. The aqueoussolution was then extracted with EtOAc (30 mL) and washed with H₂O andbrine. After drying over Na₂SO₄ the reaction was filtered andconcentrated to afford 1-7 as an orange gum (0.82 g, 88% yield). ¹H NMR(400 MHz, CDCl₃) δ 3.94 (s, 31-1), 7.69 (s, 1H), 10.85 (br s, 1H).

Methyl 4,6-dichloro-3-methoxy-2-nitrobenzoate (2-2)

To the phenol 1-7 (10.4 g, 39.1 mmol) in HPLC grade acetone (150 mL) wasadded K₂CO₃ (15 g, 108.5 mmol) and iodomethane (5 mL, 80. 3 mmol). Thereaction was heated to 60° C. for 22 h. The flask was cooled and thenH₂O (40 mL) was added and the mixture stirred for 30 min. The volatileswere removed in vacuo and the residue was taken up in CH₂Cl₂ and H₂O.After extracting into CH₂Cl₂ (×3), the combined organic layers werewashed with H₂O, dried over Na₂SO₄, filtered and concentrated to givethe methyl ester 2-2 as an orange oil (9.92 g, 91% yield). ¹H NMR (400MHz, d6-DMSO) δ 0.95 (s, 3H), 8.22 (s, 1H).

4,6-Dichloro-3-methoxy-2-nitrobenzoic Acid (2-1)

To the methyl ester 2-2 (9.90 g, 35.3 mmol) in MeOH (150 mL) was added2N NaOH (150 mL) and the reaction was heated to reflux for 2.5 h.Methanol was removed in vacuo and the mixture was extracted with EtOAc.The aqueous layer was acidified to pH 2 with a concentrated solution ofHCl and then extracted into EtOAc (×3). The combined organic layers werewashed with brine, dried over Na₂SO₄, filtered and concentrated toprovide the acid 2-1 as an orange solid (8.84 g, 94% yield). ¹H NMR (400MHz, d6-DMSO) δ 3.83 (s, 311), 3.95 (s, 3H), 8.23 (s, 1H).

Preparation of F4095 4,6-Dichloro-3-methoxy-N-propyl-2-nitrobenzamide(4-5)

The acid 2-2 (1.41 g, 5.3 mmol) was heated to reflux in thionyl chloride(20 mL) for 2 h. After cooling, the volatiles were removed in vacuo andthe residue was azeotroped with toluene. The resulting acid chloride wasdried at high vacuum and then dissolved in anhydrous CH₂Cl₂ (50 mL),cooled to 0° C. then treated with propylamine (2.45 mL, 29.9 mmol). Thereaction was warmed to room temperature for 2 h. The solvent was removedin vacuo and the residue was taken up in EtOAc and brine. The aqueouslayer was extracted three times into EtOAc and the combined organiclayers were washed with H₂O, dried over Na₂SO₄, filtered andconcentrated to provide the amide in which R is propyl 4-5 as a lightorange solid. (1.5 g, 92% yield). ¹H NMR (400 MHz, d6-DMSO) δ 0.91 (t,J=7.0 Hz, 3H), 1.40 (q, J=7.0 Hz, 2H), 3.07 (q, J=7.0 Hz, 2H), 3.84 (s,3H), 8.13 (s, 1H), 8.84 (br s, 1H).

4,6-Dichloro-3-methoxy-N-propyl-2-nitrobenzamide (4-6)

The nitro compound 4-5 in which R is propyl (1.50 g, 4.88 mmol) inglacial AcOH (50 mL) was heated to 80° C. with Fe powder (1.21 g, 21.7mmol) for 1 h. The reaction was filtered through celite, washed withEtOAc (×3) and concentrated. The residue was diluted with EtOAc and sat.aq. NaHCO₃ and the aqueous layer was extracted with EtOAc (×3). Thecombined organic layers were washed with H₂O, dried over Na₂SO₄,filtered and concentrated to provide the amine 4-6 as a light brown oil(1.21 g, 89% yield). ¹H NMR (400 MHz, d6-DMSO) δ 0.94 (t, J=7.2 Hz, 3H),1.49 (q, J=7.2 Hz, 2H), 3.18 (q, J=7.2 Hz, 2H), 3.82 (s, 3H), 5.22 (brs, 2H), 6.77 (s, 1H), 8.42 (t, J=7.2 Hz, 1H).

5,7-Dichloro-2-(chloromethyl)-8-methoxy-3-propylquinazolin-4(3H)-one(4-7)

Amine 4-6 in which R is propyl (1.18 g, 4.26 mmol) in glacial AcOH (40mL) was treated with chloroacetyl chloride (0.85 mL, 10.6 mmol) with orwithout the addition of concentrated sulfuric acid (0.6 eq) and thereaction was heated to reflux under N₂ for 4 h. Additional chloroacetylchloride was added (0.85 mL) and the reaction was heated until no morestarting material was observed by TLC. The reaction was cooled then thepH was adjusted to 5 using 2N NaOH. The reaction was extracted intoCH₂Cl₂ (×3). The combined organic layers were dried over Na₂SO₄,filtered, concentrated and purified by flash chromatography eluting with10% and then 15% EtOAc/petroleum ether 40°-60° C. to afford thechloromethyl compound 4-7 in which R is propyl as an oil (501 mg, 35%yield). ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (t, J=7.4 Hz, 3H), 1.64 (q,J=7.4 Hz, 2H), 3.97 (t, J=7.4 Hz, 211), 3.99 (s, 3H), 4.91 (s, 2H), 7.77(s, 1H).

5,7-Dichloro-2-((methylamino)methyl)-8-methoxy-3-propylquinazolin-4(3H)-one(4-8)

The chloromethyl compound 4-7 in which R is propyl (280 mg, 0.834 mmol)in anhydrous CH₂Cl₂ (8 mL) was cooled to 0° C. and methylamine solutionin ethanol (2.0 mL, 33% wt) was added dropwise to the solution. Thereaction was allowed to warm to room temperature overnight thenvolatiles were remove in vacuo. The residue was taken up in CH₂Cl₂ andsat. aq. NaHCO₃ then extracted with CH₂Cl₂ (×2). Combined organic layerswere washed with H₂O, dried over Na₂SO₄, filtered and concentrated toafford the amine 4-8 in which R is propyl and R′ and R″ are hydrogen(220 mg, 80% yield) as an oil. ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (t,J=7.2 Hz, 3H), 1.62 (q, J=7.2 Hz, 3H), 2.36 (s, 3H), 3.80 (s, 2H), 3.98(obscured triplet, J=7.2 Hz, 2H), 3.99 (s, 3H), 7.63 (s, 1H).

5,7-Dichloro-8-hydroxy-2-((methylamino)methyl)-3-propylquinazolin-4(3H)-one(F4095)

Methyl ether 4-8 (220 mg, 0.67 mmol) in CH₂Cl₂ (8 mL) was cooled to 0°C. and then BBr₃ (158 μL, 1.64 mmol) was added dropwise to the reaction.After warming to room temperature overnight the reaction was quenchedcarefully with MeOH at 0° C. The solution was concentrated and thenmethanol was added again. This procedure was repeated several times. NMRof the crude material revealed starting material (approx 15%) togetherwith the desired product. The crude material was redissolved inanhydrous CH₂Cl₂ (8 mL) and treated dropwise with BBr₃ (200 mL). Thereaction was warmed to room temperature overnight then the above work-upprocedure was employed to give a the crude product. Trituration withMeOH (1 mL)/ether (20 mL) and filtration provided the desired targetcompound 1095 (158 mg, 57% yield). ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (t,J=7.2 Hz, 3H), 1.58 (q, J=7.2 Hz, 2H), 2.74 (s, 3H), 3.90 (t, J=7.2 Hz,2H), 4.51 (s, 2H), 7.59 (s, 1H), 8.88 (s, 2H), 10.19 (s, 1H). MS m/z316.2, 318.2 [M+H]⁺.

TABLE 2 Compounds prepared according to Example 2 (Scheme 2) CompoundStructure MW ¹H NMR Mass Spec. F4161

Parent: 288.13 HBr Salt: (400 MHz, d6-DMSO) δ 2.44 (s, 3H), 3.37 (s,3H), 4.51 (t, J = 4.8 Hz, 2H), 7.60 (s, 1H), 8.99 (br s, 2H), 10.19 (s,1H) m/z 288.1, 290.1 [M + H]⁺ F4473

Parent: 330.21 HCl* Salt: 366.67 ¹H NMR (400 MHz, d6-DMSO) δ 1.23 (m,6H), 3.22 (s, 4H), 3.40 (s, 3H), 4.63 (s, 2H), 7.61 (s, 1H), 9.89 (bs,1H), 10.65 (s, 1H) m/z 330.1, 332.1 [M + H]⁺ F4475

Parent: 342.23 HBr Salt: 423.13 ¹H NMR (400 MHz, d6-DMSO) δ 1.40-1.90(m, 6H), 3.18 (m, 2H), 3.40 (s, 3H), 3.57 (m, 2H), 4.70 (s, 2H), 7.61(s, 1H), 9.09 (bs, 1H), 10.19 (s, 1H) m/z 342.1, 344.1 [M + H]⁺ F4477

Parent: 316.19 HBr Salt: 397.1  ¹H NMR (400 MHz, d6-DMSO) δ 0.97 (t, J =7.9 Hz, 3H), 1.65 (m, 2H), 3.00 (m, 2H), 3.40 (s, 3H), 4.49 (s, 2H),7.62 (s, 1H), 9.86 (bs, 1H), 10.24 (s, 1H) m/z 316.0, 318.0 [M + H]⁺F4483

Parent: 356.25 HBr Salt 437.16 ¹H NMR (400 MHz, d6-DMSO) δ 1.20 (t, J =7.6 Hz, 3H) 1.40-1.90 (m, 6H), 3.17 (m, 2H), 3.56 (m, 2H), 3.88 (q, J =7.6 Hz, 2H), 4.71 (s, 2H), 7.62 (s, 1H), 9.05 (bs, 1H), 10.23 (s, 1H)m/z 356.3, 358.1 [M + H]⁺ F4486

Parent: 302.16 HBr Salt: 363.07 ¹H NMR (400 MHz, d6-DMSO) δ 1.24 (t, J =6.8 Hz, 3H), 2.76 (s, 3H), 3.94 (q, J = 6.8 Hz, 2H), 4.58 (s, 2H), 8.98(br s, 2H), 10.25 (s, 1H). m/z 302.2, 304.2 [M + H]⁺ F4487

Parent: 410.28 HBr Salt: 491.18 ¹H NMR (400 MHz, d6-DMSO) δ 1.20 (t, J =7.8 Hz, 3H), 2.93 (s, 3H), 3.88 (m, 2H), 4.55 (m, 2H), 4.76 (s, 2H),7.23 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.65 (s, 1H), 8.79(bs, 1H), 10.21 (s, 1H) m/z 410.1, 412.2 [M + H]⁺ F4492

Parent: 370.27 HBr Salt: 451.19 ¹H NMR (400 MHz, d6-DMSO) δ 0.97 (t, J =7.0 Hz, 3H), 1.43 (m, 1H), 1.61 (m, 2H), 1.75 (m, 1H), 1.86 (m, 4H),3.22 (m, 2H), 3.59 (m, 2H), 3.85 (m, 2H), 4.78 (d, J = 4.0 Hz, 2H), 7.63(s, 1H), 9.18 (br s, 1H), 10.25 (s, 1H). m/z 370.1, 372.1 [M + H]⁺ F4495

Parent: 316.19 HBr Salt: 397.10 ¹H NMR (400 MHz, d6-DMSO) δ 1.20 (t, J =7.9 Hz, 3H), 1.31 (t, J = 7.9 Hz, 3H), 3.15 (m, 2H), 3.93 (m, 2H), 4.49(s, 2H), 7.62 (s, 1H), 8.79 (s, 2H), 10.30 (s, 1H) m/z 316.2, 318.2 [M +H]⁺ F4496

Parent: 302.16 HBr Salt: 383.07 ¹H NMR (400 MHz, d6-DMSO) δ 1.31 (t, J =6.8 Hz, 3H), 3.14 (m, 2H), 3.44 (s, 3H), 4.54 (s, 2H), 7.65 (s, 1H),8.95 (s, 2H), 10.30 (s, 1H). m/z 302.2, 304.2 [M + H]⁺ F4473

Parent: 330.22 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 1.23 (m,6H), 3.31 (m, 4H), 3.39 (s, 3H), 4.62 (s, 2H), 7.64 (s, 1H), 9.83 (bs,1H), 10.65 (s, 1H). m/z 330.1 [M + H]⁺, 332.1 F4551

Parent: 358.26 HBr Salt: 439.17 ¹H NMR (400 MHz, d6-DMSO) δ 0.89 (m,6H), 1.72 (m, 4H), 3.25 (m,4H), 3.42 (s, 3H), 4.77 (s, 2H), 7.64 (s,1H), 9.11 (bs, 1), 10.40 (s, 1H). m/z 358.1 [M + H]⁺, 360.1 F4549

Parent: 330.21 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (t, J =7.6 Hz, 3H), 1.23 (t, J = 6.8 Hz, 3H), 1.74 (m, 2H), 3.06 (m, 2H), 3.96(m, 2H), 4.56 (s, 2H), 7.65 (s, 1H), 8.94 (bs, 1H), 10.31 (s, 1H). m/z330.2 [M + H]⁺, 332.2 F4550

Parent: 372.30 HBr Salt: 453.20 ¹H NMR (400 MHz, d6-DMSO) δ 0.88 (t, J =7.2 Hz, 6H), 1.25 (m, 3H), 1.72 (m, 4H), 3.28 (m, 4H), 3.95 (d, J = 7.2Hz, 2H), 4.78 (s, 2H), 7.67 (s, 1H), 9.16 (bs, 1H), 10.39 (s, 1H). m/z372.1 [M + H]⁺, 374.1 F4530

Parent: 330.21 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (t, J =6.8 Hz, 3H), 1.63 (m, 2H), 3.00 (3, 6H), 3.80 (m, 2H), 4.76 (s, 2H),7.66 (s, 1H), 9.44 (bs, 1H), 10.23 (s, 1H). m/z 328.3 [M + H]⁺, 330.3F4540

Parent: 330.21 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 0.94 (t, J =6.8 Hz, 3H), 1.14 (t, J = 6.9 Hz, 3H), 1.49 (m, 2H), 3.17 (q, J = 6.8Hz, 2H), 3.30 (m, 2H), 4.52 (s, 2H), 7.10 (s, 1H), 8.05 (br s, 1H), 8.18(s, 1H). m/z 330.3 [M + H]⁺, 332.3 F4541

Parent: 357.26 HBr Salt: 439.17 ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (m,3H), 1.28 (m, 6H), 1.64 (m, 2H), 3.33 (m, 4H), 3.86 (m, 2H), 4.71 (m,2H), 7.67 (s, 1H), 9.14 (bs, 1H), 10.41 (s, 1H). m/z 358.3 [M + H]⁺,360.3 F4542

Parent: 344.23 HBr Salt: 425.14 ¹H NMR (400 MHz, d6-DMSO) δ 0.94 (m,6H), 1.65 (m, 2H), 1.74 (m, 2H), 3.07 (m, 3H), 3.83 (m, 2H), 3.86 (m,2H), 4.54 (s, 2H), 7.66 (s, 1H), 9.00 (s, 2H), 10.33 (s, 1H). m/z 344.3[M + H]⁺, 346.3 F4543

Parent: 386.32 HBr Salt: 467.23 ¹H NMR (400 MHz, d6-DMSO) δ 0.89 (t, J =7.2 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H), 1.64 (q, J = 7.6 Hz, 2H), 1.71(q, J = 7.6 Hz, 2H), 3.27 (m, 5H), 3.84 (t, J = 7.2 Hz, 2H), 4.76 (s,1H), 4.78 (s, 1H), 7.66 (s, 1H), 9.14 (s, 2H), 10.39 (s, 1H). m/z 386.1[M + H]⁺, 388.1 F4544

Parent: 330.22 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 0.91 (s,6H), 2.07 (m, 1H), 2.75 (s, 3H), 3.79 (d, J = 6.0 Hz, 2H), 4.52 (s, 2H),7.66 (s, 1H), 8.93 (s, 2H), 10.30 (s, 1H). m/z 330.3 [M + H]⁺, 332.3F4545

Parent: 344.24 HBr Salt: 425.15 ¹H NMR (400 MHz, d6-DMSO) δ 0.91 (s,6H), 1.30 (t, J = 7.2 Hz, 3H), 2.07 (m, 1H), 3.18 (m, 2H), 3.81 (d, J =7.6 Hz, 2H), 4.51 (s, 2H), 7.66 (s, 1H), 8.90 (s, 2H), 10.35 (s, 1H).m/z 344.3 [M + H]⁺, 346.3 F4546

Parent: 372.29 HBr Salt 453.20 ¹H NMR (400 MHz, d6-DMSO) δ 0.91 (m, 6H),1.28 (t, J = 6.8 Hz, 6H), 2.08 (m, 1H), 3.92 (m, 4H), 3.81 (d, J = 7.6Hz, 2H), 4.70 (s, 1H), 4.71 (⁴J = 0.4 Hz, 1H), 7.66 (s, 1H), 9.11 (s,2H), 10.40 (s, 1H). m/z 372.3 [M + H]⁺, 374.3 F4547

Parent: 358.27 HBr Salt: 439.18 ¹H NMR (400 MHz, d6-DMSO) δ 0.90 (m,6H), 1.28 (t, J = 7.2, 3H), 1.74 (m, 2H), 2.06 (m, 1H), 3.06 (m, 2H),3.81 (d, J = 8.0 Hz, 2H), 4.52 (s, 1H), 7.66 (s, 1H), 9.14 (s, 2H),10.35 (s, 1H). m/z 358.3 [M + H]⁺, 360.3 F4548

Parent: 400.35 HBr Salt: 481.25 ¹H NMR (400 MHz, d6-DMSO) δ 0.87-0.92(m, 12H), 1.71 (m, 4H), 2.07 (m, 1H), 3.28 (m, 4H), 3.80 (d, J = 7.6 Hz,2H), 4.75 (⁴J = 4.4 Hz, 1H), 7.66 (s, 1H), 9.19 (s, 2H), 10.40 (s, 1H).m/z 400.4 [M + H]⁺, 402.4 F4553

Parent: 344.24 HBr Salt: 425.15 ¹H NMR (400 MHz, d6-DMSO) δ 0.95 (m,3H), 1.58 (s, 3H), 1.59 (s, 3H), 1.65 (m, 2H), 3.11 (m, 2H), 4.43 (m,1H), 4.71 (s, 2H), 7.62 (s, 1H), 8.99 (bs, 1H), 10.45 (s, 1H). m/z 344.1[M + H]⁺, 346.1 F4554

Parent: 330.21 HBr Salt: 411.12 ¹H NMR (400 MHz, d6-DMSO) δ 1.25 (m,3H), 1.55 (s, 3H), 1.57 (s, 3H), 3.17 (m, 2H), 3.11 (m, 2H), 4.42 (m,1H), 4.73 (s, 2H), 7.82 (s, 1H), 8.78 (bs, 1H), 10.30 (s, 1H). m/z 330.3[M + H]⁺, 332.3 F4555

Parent: 386.32 HBr Salt: 467.23 ¹H NMR (400 MHz, d6-DMSO) δ 0.93 (m,6H), 1.57 (s, 3H), 1.58 (s, 3H), 1.74 (m, 4H), 3.17 (m, 4H), 3.11 (m,2H), 4.35 (m, 1H), 4.76 (s, 2H), 7.67 (s, 1H), 8.90 (bs, 1H), 10.33 (s,1H). m/z 386.1 [M + H]⁺, 388.1 F4552

Parent: 344.24 HBr Salt: 425.15 ¹H NMR (400 MHz, d6-DMSO) δ 0.92 (t, J =6.81Hz, 3H), 1.32 (m, 2H), 1.74 (m, 2H), 2.98 (s, 3H), 3.31 (m, 2H),3.39 (s, 3H), 4.68 (dd, ²J = 12.0, ⁴J = 5.6 Hz, 1H), 4.82 (d, ²J = 14.4Hz, 1H), 7.66 (s, 1H), 9.26 (bs, 1H), 10.27 (s, 1H). m/z 344.1 [M + H]⁺,346.1 F4582

Parent: 316.18 HBr Salt: 397.10 ¹H NMR (400 MHz, d6-DMSO) δ 1.33 (t, J =7.2 Hz, 3H), 2.96 (d, ⁴J = 4.4 Hz, 3H), 3.39 (m, 2H), 3.41, (s, 3H),4.68 (dd, ²J = 16.8, ⁴J = 5.6 Hz, 1H), 4.82 (d, ²J = 17.6 Hz, 1H), 7.66(s, 1H), 9.25 (bs, 1H), 10.27 (s, 1H). m/z 316.2, 318.2 [M + H]⁺ F4A

Parent: 316.18 F4B

Parent: 320.21 F4C

Parent: 358.26 *Same method as for the other compounds. Final compoundwas neutralized with sat. aq. NaHCO₃ and converted to the HCl salt.

Example 3 Scheme 3

2,3-Disubstituted 8-hydroxy-3H-quinazolin-4-ones 4-9 can also besynthesised by the route depicted in Scheme 3. Thus, the methyl ester2-2 (prepared according to Scheme 2 shown in Example 2) is reduced withiron powder in acetic acid to give aniline 2-3. Hydrolysis to the acid2-4 then amide formation via the acid chloride affords 4-6. Dehydrativecyclisation is achieved by the action of refluxing chloroacetyl chloridein acetic acid to afford chloromethyl intermediate 4-7. Amination,followed by deprotection with either BBr₃ or boiling hydrobromic acidgenerates the desired 2,3-disubstituted quinazolinone 4-9.

Methyl 2-amino-4,6-dichloro-3-methoxybenzoate (2-3)

Iron powder (18.2 g, 330 mmol) was added to a solution of methyl ester2-2 (13.3 g, 480 mmol) in glacial acetic acid (120 mL). The mixture wasstirred at 55° C. for 1.5 h and then filtered hot through celite,washing with ethyl acetate. The filtrate was concentrated then ethylacetate and sat. aq. NaCO₃ were added. The organic layer was isolated,washed with H₂O, dried over K₂CO₃ and concentrated to give 2-3 as anoff-white solid (11.6 g, 97% yield). ¹H NMR (CDCl₃, 400 MHz) δ 3.79 (s,3H), 3.89 (s, 3H), 6.71 (s, 1H).

2-Amino-4,6-dichloro-3-methoxybenzoic Acid (2-4)

To a stirred solution of aniline 2-3 (11.5 g, 460 mmol) in methanol (250mL) and H₂O (70 mL) was added 2N NaOH (25 mL). The reaction mixture washeated under reflux for 1 h, more 2N NaOH (25 mL) was added and themixture was heated under reflux for a further 1 h. The solution wascooled and methanol was removed in vacuo. The mixture was dissolved inH₂O and extracted with ethyl acetate. The aqueous layer was acidified topH 2 with conc. HCl. and then extracted into ethyl acetate (×3). Thecombined extracts were washed with brine, dried over Na₂SO₄, filteredand concentrated to provide the acid 2-4 as a beige solid (10.4 g, 95%yield). ¹H NMR (MeOD, 400 MHz) δ 3.80 (s, 3H), 6.70 (s, 1H).

Preparation of F4269 2-Amino-4,6-dichloro-3-methoxy-N-methylbenzamide

To a mixture of the acid (3.13 g, 13.3 mmol) in toluene (22 mL) cooledto 0° C. was added thionyl chloride (3.9 mL, 53.0 mmol). The mixture washeated to reflux for 2 h and the resulting solution was concentrated todryness. The acid chloride was dissolved in anhydrous CH₂Cl₂ (22 mL) andcooled to 0° C. Methylamine (15 mL, 8.0M solution in absolute ethanol,120 mmol) was added and the reaction was allowed to warm to roomtemperature overnight. The mixture was concentrated and purified byflash chromatography eluting with 30%-60% ethyl acetate/petroleum ether40-60° C. to provide the product as brown oil (2.43 g, 74% yield). ¹HNMR (d6-DMSO, 400 MHz) δ 2.72 (t, J=4.0 Hz, 3H), 3.68 (s, 3H), 5.33 (s,2H), 6.72 (s, 1H), 8.37 (d, J=4.0 Hz, 1H).

5,7-dichloro-2-(chloromethyl)-8-methoxy-3-methylquinazolin-4(3H)-one

To a solution of the amide (2.43 g, 9.76 mmol) in acetic acid (39 mL)was added chloroacetyl chloride (4.86 mL, 61 mmol) and the reaction washeated to reflux for 6 h then cooled to room temperature. The reactionwas concentrated and then purified by flash chromatography eluting with15%-40% ethyl acetate/petroleum ether 40-60° C. to provide the chlorideas a solid (878 mg, 57% yield). ¹H NMR (d6-DMSO, 400 MHz) δ 3.54 (s,3H), 4.00 (s, 3H), 4.91 (s, 2H), 7.74 (s, 1H).

5,7-Dichloro-2-((dimethylamino)methyl)-8-methoxy-3-methylquinazolin-4(3H)-one

To a solution of the chloromethyl compound (500 mg, 1.63 mmol) at 0° C.was added dimethylamine (12 mL, 2.0M, 24 mmol). The reaction was warmedto room temperature and stirred for 3 days and concentrated. The crudeproduct was purified by flash chromatography eluting with 70%-80%-100%ethyl acetate/petroleum ether 40-60° C. to afford the amine as a yellowsolid (391 mg, 76% yield). ¹H NMR (d6-DMSO, 400 MHz) δ 2.26 (s, 6H),3.56 (s, 2H), 3.58 (s, 3H), 3.97 (s, 3H), 7.68 (s, 1H).

5,7-Dichloro-2-(dimethylamino)methyl-8-hydroxy-3-methylquinazolin-4(3H)-one(PB1269)

To a mixture of the methoxy derivative (391 mg, 1.24 mmol) in anhydrousCH₂Cl₂ (6 mL) at 0° C. was added BBr₃ (234 mL, 2.48 mmol). The mixturewas stirred at room temperature for 36 h after which it was cooled to 0°C. and the reaction was cautiously quenched with methanol. The solutionwas concentrated and then methanol was added again. This procedure wasrepeated several times. To the crude product was added ether and a fewdrops of methanol to precipitate F4269 as a cream solid that wascollected by filtration and dried (340 mg, 72% yield). δ ¹H NMR (400MHz, d6-DMSO) δ 2.95 (s, 3H), 2.96 (s, 3H), 3.33 (s, 3H), 4.67 (d, J=5.2Hz, 2H), 7.62 (s, 1H), 9.37 (br s, 1H), 10.2 (s, 1H). m/z 315.1, 316.2[M+H]⁺

Example 4 Scheme 4

2,3-Disubstituted 8-hydroxy-3H-quinazolin-4-ones 4-9 can also beprepared by the route depicted in Scheme 4. Nitro acid 1-6 (preparedaccording to Scheme 1 shown in Example 1) was reduced to aniline 1-8with iron powder and acetic acid. Acylation of 1-8 with chloroacetylchloride provides the amide 1-9. One pot amide formation followed bydehydrative cyclisation to chloride 1-10 is achieved by the action ofPCl₃ and an amine in refluxing toluene. After purification, amination ofthe chloromethyl compound 1-10 affords target compound F4271.

2-Amino-4,6-dichloro-3-hydroxybenzoic Acid (1-8)

A mixture of 4,6-dichloro-3-hydroxy-2-nitrobenzoic 1-6 (700 mg, 2.78mmol), Fe powder (400 mg, 7.16 mmol) and glacial acetic acid (13 mL) washeated at 80° C. for 50 min, cooled and the solids filtered off. Thefiltrate was concentrated to a brown solid. Purification by flashchromatography eluting with 1% AcOH/EtOAc to 3% AcOH/EtOAc afforded2-amino-4,6-dichloro-3-hydroxybenzoic acid (1-8) as a light brown solid(582 mg, 94%). ¹H NMR (d6-DMSO, 400 MHz) δ. 6.68 (s, 1H).

4,6-Dichloro-2-(2-chloroacetamido)-3-hydroxybenzoic acid (1-9)

To a mixture of the acid (1.0 g, 4.50 mmol) in anhydrous CH₂Cl₂ (22 mL)was added chloroacetyl chloride (1.4 mL, 18 mmol) at 0° C. The reactionwas warmed to room temperature for 1 h and then concentrated to affordan orange oil. ¹H NMR (d6-DMSO, 400 MHz) δ 4.18 (s, 2H), 7.56 (s, 1H),9.83 (s, 1H), 10.22 (s, 1H).

5,7-Dichloro-2-(chloromethyl)-8-hydroxy-3-isobutylquinazolin-4(3H)-one

To a mixture of the acid (500 mg, 1.68 mmol) in anhydrous toluene (8 mL)was added PCl₃ (293 pt, 3.36 mmol) then isobutylamine (250 pt, 2.52mmol). The reaction was heated to reflux for 2.5 h and then cooled toroom temperature and concentrated. H₂O was added followed by sat. aq.NaHCO₃ until the pH was 7. The mixture was extracted in EtOAc (×3) andthe combined organic layers were washed with brine, dried over Na₂SO₄,filtered, concentrated and purified by flash chromatography eluting with20% EtOAc/petroleum ether 40°-60° C. to provide the chloromethylderivative (71 mg, 13% yield). ¹H NMR (d6-DMSO, 400 MHz) δ 0.90 (d,J=6.8 Hz, 1H), 2.17 (m, 1H), 3.93 (d, J=7.6 Hz, 2H), 4.83 (s, 2H), 7.63(s, 1H), 10.47 (s, 1H).

5,7-Dichloro-2-((dimethylamino)methyl)-8-hydroxy-3-isobutylquinazolin-4(3H)-one(F4271)

To a solution of the chloromethyl compound (50 mg, 0.149 mmol) inanhydrous CH₂Cl₂ (2 mL) at 0° C. was added dimethylamine (3 mL, 2.0Msolution in MeOH, 6 mmol). The reaction was warmed to room temperatureovernight then concentrated to dryness. The residue was taken up in MeOH(2 mL) and conc. HCl (0.5 mL) and then volatiles were removed in vacuo.Trituration with MeOH (few drops) and ether provided the desired amine(PB1271) as an off-white solid (19 mg, 33%). NMR (400 MHz, d6-DMSO) δ0.48 (m, 4H), 1.19 (t, J=4.80 Hz, 1H), 2.94 (s, 3H), 2.95 (s, 3H), 3.85(d, J=6.8 Hz, 2H), 4.86 (d, J=4.80 Hz, 2H), 7.63 (s, 1H), 8.68 (br s,1H), 10.64 (s, 1H), 10.65 (br s, 1H). MS m/z 344.3, 346.3 [M+H]⁺.

Example 5 Assessment of Compounds of Formula I

The following Assays were used in the assessment of the compounds offormula I for suitability for use in the methods of the invention.

Assay 1. H₂O₂ Inhibition Assay.

This fluoresence assay evaluates the ability of a test compound toinhibit the generation of hydrogen peroxide (H₂O₂) by iron in thepresence of a reducing substrate such as ascorbic acid. In the assay,iron in the form of FeCl₃ or copper is allowed to react with ascorbicacid by incubating for 1 hr at 37° C. in the presence of the fluorescingcompound DCF and horseradish peroxidase. H₂O₂ generated by the system isassessed by measuring the specific fluorescence profile at theexcitation and emission wavelengths of 485 and 530 nm respectively, inthe presence of increasing concentrations of test compound. Testcompounds are ranked according to their capacity to inhibit H₂O₂generated by the system where lower values reflect greater ability toinhibit H₂O₂ production.

Assay 2 (a). Primary Neuronal Cells Neurotoxicity Assays

Cortical cultures were prepared as previously described (White et al.,1998). Embryonic day 14 BL6Jx129sv mouse cortices were removed,dissected free of meninges and dissociated in 0.025% (wt/vol) trypsin.Dissociated cells were plated in 48 well culture plates at a density of2×10⁶ cells/mL in MEM with 25% (vol/vol) FCS and 5% (vol/vol) HS andincubated at 37° C., 2 hrs. Media was then replaced with Neurobasalmedia (Invitrogen Life Technologies) and B27 supplements (InvitrogenLife Technologies). Cultures were maintained at 37° C. in 5% CO₂. Priorto experimentation, the culture medium was replaced with Neurobasalmedia and B27 minus antioxidants (Invitrogen Life Technologies).

Neuronal cortical cells were cultured for five days as per Assay 2 in NBmedia and B27 supplement.

On day six the test compounds were added to the neuronal cell culturesin NB media and B27 supplement minus antioxidants.

Test compounds were dissolved in 100% DMSO to a concentration of 2.5 mM(10 mM if excess compound was weighed out per vial—then diluted to 2.5mM). 2.5 mM stock solution was serially diluted 1 in 10 to give workingsolutions of 250 uM, 25 uM, 2.5 uM. Test compounds were not addeddirectly to cells, instead they were added to a 48 well ‘Drug Plate’ ascomprised below:

-   -   Preparation of “Drug Plate”:

To a 48 well plate add:

Well 1: 576 ul NB+B27 (no antioxidant)*+24 ul 2.5 uM test compound

Well 2: 576 ul NB+B27 (no antioxidant)+24 μl 25 uM test compound

Well 3: 576 ul NB+B27 (no antioxidant)+24 ul 250 uM test compound

Well 4: 576 ul NB+B27 (no antioxidant)+24 ul 2.5 uM test compound

Well 5: 576 ul NB+B27 (no antioxidant)+24 ul 25 uM test compound

Well 6: 576 ul NB+B27 (no antioxidant)+24 ul 250 uM test compound

Well 7: 576 ul NB+B27 (no antioxidant)+24 ul test compound diluent**

Well 8: 600 ul NB+B27 (no antioxidant)

The Drug Plate was incubated at 37° C. for 15 mins. 200 ul of each wellwas added in triplicate to the corresponding cell plate. The cell platewas incubated at 37° C., for 4 days, (2 compounds are tested on eachplate of cells).

*NB media and B27 (no antioxidants),

**PBT diluent 10% DMSO in NB+1327 (no antioxidants)

On completion of the assay, 1/10 volume MTS was added per well of plate(ie 25 ul/250 ul). The plates were incubated at 37° C. for 2 hrs, andthen absorbance was read at 560 nm.

Assay 3. Metal Uptake Assay

M17 human neuroblastoma cells are plated out on 6 well plates and leftovernight. Enough cells are added to give approximately 70% confluentthe following day of the experiment. Test compounds are added to mediaand mixed with equi-molar amounts of CuCl2 solution. A=10 μM Cu+10 μMMPAC; B=10 μM Cu+10 μM MPAC.

Cells are incubated in 1 ml of media/MPAC/Cu mix for 5 hours at 37° C.At the end of the incubation the media is removed with a vacuumaspirator and 1 ml of PBS added to dislodge the cells. Cells are thenput into Eppendorf tubes and pelleted. The PBS is removed and theremaining cell pellets are frozen at −20 C.

The cell pellets are prepared as follows:

Received cell pellets of similar levels in 1.5 ml microfuge tubes. Added50 μl of concentrated Nitric Acid (Aristar, BDH) to each cell pellet andallowed them to digest over night. Heated the samples for 20 min at 90°C. to complete the digestion. The volume of each sample was reduced to˜45 ul after digestion. Added 1 ml of the 1% Nitric Acid diluent to eachsample. (referred to as the “preparation solution” samples).

Measurements were made using a Varian UltraMass ICPMS instrument underoperating conditions suitable for routine multi-element analysis.

The instrument was calibrated using Blank, 10, 50 and 100 ppb of acertified multi-element ICPMS standard solution (ICP-MS—CAI2-1,Accustandard) for Fe, Cu and Zn in 1% nitric acid. Used an certifiedinternal standard solution containing 100 ppb Yttrium (Y 89) as aninternal control (ICP-MS—IS-MIX1-1, Accustandard).

Assay 4. Physiochemical Properties

Polar Surface Area Calculations (PSA)

Polar surface area values were calculated using the web-based programavailable through “Molinspiration”, a package for calculation ofmolecular properties.

Turbidimetric Solubility Measurements

The solubility estimate was measured at both pH 2.0 and pH 6.5. This iswithin the pH range that can be anticipated along the proximalgastrointestinal tract in humans.

The compounds are dissolved in DMSO to appropriate concentrations andthen spiked into either 0.01 M HCl (approx. pH=2.0) or pH 6.5 isotonicphosphate buffer, the final DMSO concentration being 1%. Samples arethen analysed via Nephelometry to determine a solubility range. [as perD. Bevan and R. S. Lloyd, Anal. Chem. 2000, 72, 1781-1787].

cLog P Values

Theoretical Log P values are determined using the ACD Log P software.The values quoted have been calculated from an untrained database andrefer to the unionised species.

Assay 5. Pharmacokinetic Profile

The pharmacokinetic profile of test compounds is determined by thefollowing assay:

-   -   Intravenous infusion of test compound; 2 mg/Kg in a suitable        vehicle is administered to 2 rats and arterial blood is sampled        up to 24 hours.    -   Oral administration of test compound; 30 mg/Kg in a suitable        vehicle is administered via oral gavage to 2 rats and arterial        blood is sampled up to 24 hours.    -   Plasma concentrations of test compound are determined by        suitable analytical method.

Calculations:

${CL}_{total} = \frac{{Dose}_{IV}}{{AUC}_{IV}}$$V_{d\;\beta} = \frac{{CL}_{total}}{\beta}$${{BA}(\%)} = \frac{{AUC}_{oral}*{Dose}_{IV}}{{AUC}_{IV}*{Dose}_{oral}}$

-   CL_(total)=total plasma clearance after IV administration-   V_(dβ)=volume of distribution during the elimination phase after IV    administration-   BA=oral bioavailability-   AUC_(IV)=area under the plasma concentration versus time profile    from time zero to infinity after IV administration-   AUC_(oral)=area under the plasma concentration versus time profile    from time zero to infinity after oral administration-   β=terminal elimination rate constant after IV administration    Assay 6. Blood Brain Barrier Penetration

Each compound tested demonstrates a permeability across a healthy BBB.

A bolus injection of each of the test compound (50 μL of a 3 mg/mLaqueous solution containing 40% propylene glycol and 10% ethanol) wasadministered by tail vein injection to male Swiss Outbred mice (5-7weeks of age). Alternatively, test compound was orally administered tomice according to standard procedures known to the skilled person.

At 5 and 60 min post-dose (n=3 mice at each time point), blood wascollected by cardiac puncture and the whole brain was removed by makingan incision through the back of the skull. Mice were anaesthetisedapproximately 3-4 min prior to blood and brain harvest with anintraperitoneal injection of ketamine and xylazine (133 mg/kg and 10mg/kg, respectively).The whole brain was placed into pre-weighed polypropylene vials andstored at −20° C. until analysis. On the day of analysis, the wholebrain was homogenised in 3 parts of water (on ice to reduce thepotential for ex vivo brain degradation) and an aliquot of the brainhomogenate and plasma was analysed for compound concentration by LCMS.Standards were prepared by spiking blank brain homogenate and bothsamples and standards were processed by adding acetonitrile to thetissue homogenate, centrifuging and injecting an aliquot of thesupernatant onto the LCMS.

To ensure complete recovery of compound from the brain, brain homogenatewas spiked with compound (in 50% acetonitrile:50% water) to a nominalconcentration of 500 ng/mL. The concentration of compound in thesupernatant was then determined by LCMS and compared to the supernatantconcentration when compound was added following precipitation withacetonitrile.

Calculations

C_(brain) = C_(brainhomogenate) − C_(brain  vasculature)C_(brain  vasculature) = C_(plasma) * V_(p)${B\text{:}P} = \frac{C_{brain}}{C_{plasma}}$${P_{app}( {{cm}\text{/}s} )} = \frac{C_{brain}}{\int_{0}^{t}{{C_{plasma}.\ {\mathbb{d}t}}*A}}$

-   C_(brain)=concentration of compound in brain parenchyma (ng/g)-   C_(brain homogenate)=concentration of compound in brain homogenate    (ng/g)-   C_(brain vasculature)=concentration of compound in brain vasculature    (ng/g)-   C_(Plasma)=concentration of compound in plasma (ng/mL)-   V_(P)=brain plasma volume (26 μL/g for male Swiss Outbred mice)-   B:P=brain-to-plasma ratio-   P_(app)=apparent permeability coefficient of compound permeating the    blood-brain barrier-   ∫₀ ^(t)c_(plasma)dt=concentration of compound in plasma from time    zero to 5 min post-dose (equivalent to the 5 min post-dose plasma    concentration, assuming no back diffusion from brain to plasma    within this time period)-   A=surface area of capillaries forming the blood-brain barrier (240    cm²/g brain weight for mouse)    Assay 7    In Vitro Metabolism in Human Liver Microsomes

Incubation Methods:

The solubility of test compounds and their recovery from the incubationmedia were confirmed prior to the metabolic assay. Metabolic stabilitywas performed by incubating test compounds individually (1 μM) at 37° C.with human liver microsomes. The metabolic reaction was initiated by theaddition of a NADPH-regenerating system (i.e. NADPH is the cofactorrequired for CYP450-mediated metabolism) and quenched at various timepoints over the incubation period by the addition of acetonitrile.Additional samples with dual co-factors, i.e. NADPH and UDPGA (theco-factor for glucuronidation), were also included in the incubation forthe qualitative assessment of the potential for glucuronide formation.The relative loss of parent compound and formation of metabolic productswas monitored by LC/MS using a Waters/Micromass LCT mass spectrometer.

Calculations:

Test compound concentration versus time data were fitted to anexponential decay function to determine the first-order rate constantfor substrate depletion. In cases where clear deviation from first-orderkinetics was evident, only the initial linear portion of the profile wasutilised to determine the degradation rate constant (k). Each substratedepletion rate constant was then used to calculate: [1] degradationhalf-life, [2] an in vitro intrinsic clearance value(CL_(int,in vitro)); [3] a predicted in vivo intrinsic clearance value(CL_(int)); and [4] a predicted in vivo hepatic extraction ratio(E_(H)).

$\begin{matrix}{t_{1\text{/}2} = \frac{\ln(2)}{k}} & \lbrack 1\rbrack \\{{CL}_{{int},\mspace{14mu}{{in}\mspace{14mu}{vitro}}} = \frac{k}{{microsomal}\mspace{14mu}{protein}\mspace{14mu}{consent}\mspace{14mu}( {0.4\mspace{14mu}{mg}\mspace{14mu}{protein}\text{/}{mL}} )}} & \lbrack 2\rbrack \\{{CL}_{int} = {{CL}_{{int},\mspace{14mu}{{in}\mspace{14mu}{vitro}}} \times \frac{{liver}\mspace{14mu}{mass}\mspace{14mu}(g)}{{body}\mspace{14mu}{weight}\mspace{14mu}({kg})} \times \frac{45\mspace{14mu}{mg}\mspace{14mu}{microsomal}\mspace{14mu}{protein}}{g\mspace{14mu}{liver}\mspace{14mu}{mass}}}} & \lbrack 3\rbrack^{*} \\{E_{H} = {\frac{{CL}_{blood}}{Q} = \frac{{CL}_{int}}{Q + {CL}_{int}}}} & \lbrack 4\rbrack^{*}\end{matrix}$

-   -   The following scaling parameters were assumed for estimating        metabolic stability parameters:

Liver mass Flow rate (Q) Species (g liver/kg body mass) (mL/min/kg)Human 25.7 20.7 45 mg microsomal protein/g liver mass was assumed

Predictions of In Vivo Hepatic Clearance and Hepatic Extraction Ratios:

The microsome-predicted hepatic extraction ratios (E_(H)) obtained basedon the relative rate of degradation of the test compound in vitro, leadto test compounds being classified as low (<0.3), intermediate(0.3-0.7), high (0.7-0.95) or very high (>0.95) extraction compounds.The assumptions underlying this classification are stated below.

Note:

The use of hepatic microsomes in the prediction of the in vivo hepaticclearance and extraction ratio is based on a number of assumptions(Obach, 1999; Drug Metab. Dispos. 27: 1350-1359):

1) Hepatic (microsomal) metabolic clearance is the major clearancemechanism for compounds in vivo;

2) NADPH-dependent oxidative metabolism predominates over othermetabolic routes (i.e. direct conjugative metabolism, reduction,hydrolysis, etc.); and,

3) Rates of metabolism and enzyme activities in vitro are trulyreflective of those that exist in vivo.

Calculations of intrinsic clearance are based on the “in vitro T₁₁₂method” (Obach, 1999), which has two further inherent assumptions:

1) The substrate concentration employed is well below the apparent K_(M)for substrate turnover; and,

2) There is no significant product inhibition, nor is there anymechanism based inactivation of enzyme.

Data should be considered within these terms of reference.

CYP450 Isoform Inhibition

Fluorescence Based Assay

Microsomes containing individual recombinant human CYP450 enzymes(Supersomes™) were incubated in the presence of a fixed concentration ofa probe substrate that forms a fluorescent metabolite uponCYP450-mediated metabolism. Varying concentrations of the test compound(i.e. potential inhibitor; 40-0.06 μM) were added to those incubations,and the IC₅₀ of test compound assessed according to the percentreduction in the extent of formation of the fluorescent metabolite asdetermined via analysis of the overall fluorescence response at a givenwavelength for each isoform.

A known inhibitor of each CYP450 isoform was included in each assay as apositive control and its IC₅₀ was compared to literature values foracceptance of the assay.

The inherent fluorescence of the test compound was examined under thespecific assay conditions for each isoform before and after thefluorometric assay to identify any compound-specific interference due tothe parent compound or metabolic products. If the backgroundfluorescence in these samples increased due to compound-specificinterference, then the CYP450 inhibition samples were quantified usingLC/MS assay. The positive control inhibitors were analysed by bothmethods (fluorescence and LC/MS detection) to confirm that the method ofquantitation did not alter the IC₅₀ value.

CYP450 Isoform Inhibition Using a Specific Substrate Approach

The present study is a “Tier 2” screen to assess potential enzymeinhibition at a test compound concentration of 20 μM using a specificsubstrate approach in human liver microsomes.

The substrate specific enzyme inhibition study relies on the formationof a metabolite that is mediated by a specific CYP450 isoform usinghuman liver microsomes. In the current study, the following reactionswere monitored to assess interactions with specific CYP450 isoforms. Aknown inhibitor of each isoform was included in each assay as a positivecontrol. The following IC₅₀ values have been reported in the literatureusing equivalent assay conditions for each CYP450 isoform:

Positive control Literature CYP450 isoform Metabolic pathway inhibitorIC₅₀ value CYP1A2 Phenacetin-O-deethylase Furafylline 1.76^(a) CYP2C9diclofenac-4′-hydroxylation Sulfaphenazole 0.27^(a) CYP2C19(S)-mephenytoin-4′hydroxylation Ticlopidine 2.7^(b) CYP2D6dextromethorphan-O-demethylation Quinidine 0.058^(a) CYP2E1Chlorzoxazone-6-hydroxylation Tranylcypromine 8.94^(a) CYP3A4*midazolam-1′-hydroxylation Ketoconazole 0.019^(a)testosterone-6β-hydroxylation 0.026^(a) *Recommended that twostructurally unrelated substrates be used for CYP3A4; Bjornsson et al.(2003) Drug. Metab. Dispos. 31: 815-832 ^(a)Walsky and Obach (2004)Drug. Metab. Dispos. 32: 647-660 ^(b)Turpeinen et al. (2004) Drug.Metab. Dispos. 32: 626-631

A single concentration (20 μM) of the test compound was incubated at 37°C. concomitantly with a specific substrate for an individual CYP isoformat ≦K_(m) (i.e. phenacetin 50 μM, diclofenac 6 μM, (S)-mephenyloin 50μM, dextromethorphan 3 μM, chlorzoxazone 20 μM, midazolam 2.5 μM andtestosterone 50 μM) in human liver microsomes at a protein concentrationof either 0.4 mg/mL (CYP1A2, CYP2C9, CYP2E1, CYP3A4 and CYP2D6) or 1.0mg/mL (CYP2C19). The reaction was initiated by the addition of anNADPH-regenerating system and was quenched by the addition ofacetonitrile prior to determining the concentration (and apparent rateof formation) of the specific metabolite by LC/MS.

The CYP450 inhibitory effect (i.e. % inhibition) of the test compound ata concentration of 20 μM was assessed according to the percent reductionin the apparent rate of formation of the specific metabolite, notingthat the maximal metabolite formation occurs in the absence ofinhibitor, Note that the IC₅₀ values for positive controls against eachCYP450 isoform were estimated based on the % inhibition of CYP450activity at two concentrations which bracketed the expected IC₅₀ value.

The IC₅₀ was deemed to be the concentration at which there was a 50%reduction in the amount of metabolite formed, relative to the maximalextent of formation.

TABLE 3 Biological Data In vitro Efficacy Profile H₂O₂ IC₅₀ (μM)^(a)Cytotox Physico-chemical Fe-DA % cf. (% viable properties Fe 0.4 μM/ at1 and Metal Parent MW/ DA 50 μM 10 uM)^(b) transport PSA ClogP F4271

0.17  57% M17 104.4, 61.5 102.3, 52.5 Metal transport 27.8% 344.34  HCl380.70  3.17  F4383

1.51  81% M17: 118.5, 103.4 102.3, 92.7 Metal transport   8% 411.33 484.25  (2•HCl salt) 3.57  F4384

2.15  59% M17: 116.3, 86.5 106.4, 73.0 Metal transport   17% 384.27 420.73  (HCl salt) 2.84  F4385

0.91  86% M17: 99.2, 97.7 105.0, 97.7 Metal transport   9% 370.28 406.74  (HCl salt) 3.63  F4386

0.33  77% M17: 101.8 105.2 99.8 91.9 101.4 111.7 107.9 106.8 Metaltransport   13% 344.24  380.70  (HCl salt) 3.18  F4387

0.46  77% M17: 104.0 19.8 92.7 20.7 95.9 35.5 100.7, 41.7 Metaltransport  111% 366.24  402.7   (HCl Salt) 3.17  F4391

0.59  97% (2 μM Fe) M17: 87.4, 57.5 95.9, 70.5 96.0 66.5 Metal transport  34% 382.28  418.74  (HCl salt) 3.88  F4392

0.57  72% Metal transport  161% 340.20  376.66  (HCl salt) 2.73  F4473

0.6   62% 97.0 91.6 99.0 106.1 Metal transport   9% 330.22  HBr Salt411.12  2.77  F4475

0.5   80% Metal transport   8% 342.23  HBr Salt: 423.13  2.906 F4477

0.22  65% Metal transport   16% 316.19  HBr Salt 397.1   2.305 F4480

0.47  46% 91.6, 30.3 106.7, 33.3 Metal transport  138% 436.31  HCl:472.77  4.60  F4483

0.33  62% 93.4, 99.7 105.5, 72.3 Metal transport   22% 356.25  HBr salt:437.16  3.44  F4486

1.93  56% 97.9, 52.2 101.4, 43.3 Metal transport   14% 302.16  HBr Salt383.07  1.776 F4487

0.76  53% Metal transport  133% 410.2786 HBr Salt 491.18  F4492

1.14  55% Metal transport   18% 370.27  HBr Salt: 451.19  3.96  F4495

0.77  45% 89.3, 47.9 112.8, 57.1 Metal transport   8% 316.189  HBr Salt:397.095  2.31  F4496

0.83  45% 102.7, 59.4 114.6, 101.2 Metal transport   7% 302.162  HBrSalt: 383.069  1.77  F4499

1.89  55% 99.1, 68.6 76.5, 78.1 Metal transport   15% 368.26  HCl salt404.72  3.32  F4530

0.29  57% 105.0, 57.6. 96.2, 41.4. Metal transport   24% 409.00  HBrsalt: 411.12  2.77  F4535

0.88  62% 87.0, 63.6. 103.0, 59.8. Metal transport   25% 410.34  HClsalt 446.80  4.80  F4536

1.0  81% 92.7, 64.9. 105.1, 91.9. 109.8, 40.9 Metal transport   24%410.34  HCl salt: 446.80  4.72  F4540

>10 122% 100.4, 83.4. 111.5, 96.5 Metal transport   14% 330.21  HBr salt411.12  4.72  F4541

0.74 107% 97.3, 76.6. 96.4, 90.8. Metal transport   13% 357.26  HBrsalt: 439.17  3.83  F4542

0.36  47% 105.8, 36.6. 102.9, 73.4. 94.3, 48.7. Metal transport   30%344.23  HBr salt: 425.14  3.36  F4543

0.50  71% 92.0, 52.6. 107.4, 61.5. Metal transport   62% 386.32  HBrsalt: 467.23  4.89  F4544

0.83 164% 84.0, 29.7. 86.9, 26.7. Metal transport   19% 330.22  HBrsalt: 411.12  2.70  F4545

0.43 148% 86.4, 36.2. 85.0, 28.0. Metal transport   40% 344.24  HBrsalt: 425.15  3.23  F4546

0.46  66% 95.6, 63.7. 92.2, 86.5. 103.3, 100.3. Metal transport   21%372.29  HBr salt: 453.20  4.23  F4547

0.50  34% 75.0, 25.2. 83.9, 26.0. Metal transport   54% 358.27  HBrsalt: 439.18  3.76  F4548

0.38  40% 96.5, 55.9. 96.0, 46.0. Metal transport   38% 400.35  HBrsalt: 481.25  5.29  F4549

0.30  40% 98.5, 44.6. 99.7, 52.1. Metal transport   22% 330.21  HBrsalt: 411.12  2.83  F4550

0.75 128% 88.7, 71.7. 94.7, 83.5 Metal transport   35% 372.30  HBr salt:453.20  4.36  F4551

0.45  56% 89.7, 50.7. 96.9, 62.5. Metal transport   30% 358.26  HBrsalt: 439.17  3.83  F4552

0.63  59% 97.8, 58.7. 99.0, 62.8. Metal transport   94% 344.24  HBrsalt: 425.15  3.30  F4553

344.24  HBr salt: 425.15  3.14  F4554

330.21  HBr salt: 411.12  2.61  F4555

386.32  HBr salt: 467.23  F4581

0.50 356.25  HBr salt: 392.71  3.22  F4582

0.55 316.19  HBr salt: 397.10  2.24  ^(a)concentration in μM of testcompound required to inhibit 50% of A_(beta) H₂O₂ production. ^(b)%inhibion of A_(beta) toxicity (average)

TABLE 4 Biological Data Bioavailability Tox- Brain to icity Plasma MousePlasma Druggability at Protein Plasma Ratio CYP450 Equilibrium 30 Com-Binding Conc. (IV) PK Studies Metabolism Isoforms Study Solubility mg/pound (%)^(a) (ng/mL)^(b) (B:P)^(c) In the Rat^(d) (Predicted E_(H))^(e)(IC₅₀)^(f) (pH 2-7)^(g) Kg^(h) F4161 Human Up to NA t_(1/2) = 6.7 hMicrosomes: Analysis by 196 μg/mL- Not 95.7 510.4 Cmax = Human <0.2LCMS: 746 μg/mL - toxic 6.7 μM Hepatocytes: CYP1A2 pH dependent Tmax =Human = IC₅₀: 25.7 uM 15 min 0.20 CYP2C91 % dose in Dog = 0.56 IC₅₀: >40uM urine = 0.1 Rat = 0.29 CYP2C19 AUC_(0-24 h) = IC₅₀: >40 uM 846.6μM/min CYP2D6 l₂₄ = 0.045-0.15 μM IC₅₀: 40 uM (substrate specificapproach) CYP3A4 IC₅₀: 13.8 uM F4267 Human Up to 3.46 at EnterohepaticMicrosomes: Substrate 393-1705 Not 99.5 1166.6 5 min recycling Human<0.31 Specific: μg/mL - toxic 2.02 at t_(1/2) = 4.9 h Hepatocytes:CYP1A2 pH dependent 60 min (oral dosing Human = IC₅₀: 15.3 uM only-300.44 CYP2C9 mg/Kg) Dog = 0.39 IC₅₀: >30 uM l₂₄ = 0.25-0.5 uM Rat = 0.45CYP2C19 IC₅₀: >30 uM CYP2D6 IC₅₀: 7.4 uM CYP3A4 IC₅₀: >30 uM F4268 MouseUp to 2.96 at Enterohepatic Microsomes: Substrate 3814-10184 Not 79.7-975.9 5 min recycling Human <0.2 specific: μg/mL - toxic 82.2 2.63 att_(1/2) = 4.3 h Hepatocytes: CYP1A2 not pH dependent Rat 60 min (oral-30Human = IC50: ~20 uM 94.5- mg/Kg) 0.28 CYP2C9 94.8 Cmax = 10 Dog = 0.49IC₅₀: >20.0 uM Human uM Rat = 0.30 CYP2C19 97.1 Tmax = 20 IC₅₀: >20.0 uMmin CYP2D6 Rel. IC₅₀: >20 uM Exposure: CYP2E1 51.9% IC₅₀: >20 uM 0.1%dose CYP3A4- in urine Midazolam l₂₄ = 0.02-0.05 uM IC₅₀: ~20 uM CYP3A4-Testosterone IC₅₀: >20 uM F4269 Mouse Up to 2.96 at EnterohepaticMicrosomes: Substrate 166-2670 Not 34.2- 492.1 5 min recycling Human<0.2 specific: μg/mL - toxic 41.8 2.12 at t_(1/2) = 5.6 h Hepatocytes:CYP1A2 24.1 uM pH dependent Rat 60 min (oral-20 Human = CYP2C9 >30 uM88.4- mg/Kg) 0.38 CYP2C19 >30 uM 92.6 Cmax = 3.3 Dog = 0.54 CYP2D6 >30uM Human uM Rat = 0.35 CYP3A4- 94.1- Tmax = 22.5 Midazolam >30 uM 94.6min CYP3A4- AUC_(0-24 h) Testosterone >30 uM (min*umol/L) = 330.29 0.1%dose in urine l₂₄ = 0.01-0.03 uM F4385 Human Up to Oral EnterohepaticMicrosomes: Substrate 683-1662 Not 98.7- 9074 dosing: recycling Human<0.2 specific: μg/mL - toxic 99.2 0.31 at t_(1/2) = 14.7 h Hepatocytes:CYP1A2 13.4 uM pH dependent 30 min, (oral-20 Human = CYP2C9 >30 uM 0.42at mg/Kg) 0.55 CYP2C19 >30 uM 240 min Cmax = 7.3 Dog = 0.37 CYP2D6 5.1uM uM Rat = 0.35 CYP3A4- Tmax = 30 min Midazolam 21.5 uM AUC_(0-24 h)CYP3A4- (min*umol/L) = Testosterone >30 uM 3124.7 0.05% dose in urinel₂₄ = 0.5-1.5 u F4386 NA Up to NA NA NA NA NA Not 3294.1 toxic F4387 NAUp to 0.65 at NA Microsomes: NA NA Not 942.7 5 min Human = toxic 0.29 at0.75 60 min F4391 NA Up to NA NA NA NA NA Not 17255.9 toxic F4473 NA Upto NA NA NA NA NA Not 1356.2 toxic F4483 NA Up to NA NA NA NA NA Not1585.2 toxic F4486 NA Up to Oral NA NA NA NA Not 1115 dosing: toxic 0.23at 30 min, 0.22 at 240 min F4495 Human Up to Oral EnterohepaticMicrosomes: Substrate 2227-4727 Not 97.7- 8935 dosing: recycling Human<0.2 specific: μg/mL- toxic 97.6 0.65 at t_(1/2) = 4.1 h Hepatocytes:CYP1A2 13.7 uM not pH dependent 30 min, (oral-20 Human = CYP2C9 >30 uM0.64 at mg/Kg) 0.22 CYP2C19 >30 uM 240 min Cmax = 56.7 Dog = 0.56 CYP2D69.5 uM uM Rat = 0.35 CYP3A4- Tmax = 30 min Midazolam >30 uM AUC_(0-24 h)CYP3A4- (min*umol/L) = Testosterone >30 uM 5218.4 0.18% dose in urinel₂₄ = 0.05-0.3 uM F4496 Human Up to Oral Enterohepatic Microsomes:Substrate 224-1504 Not 91.8- 3330.8 dosing: recycling Human <0.2specific: μg/mL- toxic 93-1 1.42 at t_(1/2) = 6.1 h Hepatocytes: CYP1A222 uM not pH dependent 30 min, (oral-20 Human = CYP2C9 >30 uM 1.97 atmg/Kg) 0.43 CYP2C19 >30 uM 240 min Cmax = 7.61 uM Dog = 0.66 CYP2D6 >30uM Tmax = 45 min Rat = 0.35 CYP3A4- AUC_(0-24 h) Midazolam: 4.4 uM(min*umol/L) = CYP3A4- 1069.3 Testosterone 8.6 uM 0.19% dose in urinel₂₄= 0.02-0.1 uM

Example 6 Parkinson's Disease In Vivo Technique: 6-OHDA Model

Preparation of the toxin 6-OHDA (1.65 mg/mL, Sigma Cat# H-4381):

6-OHDA toxin is dissolved in ascorbic acid solution (0.2 mg/mL in dH₂O),store in dark on ice. To increase the speed of anaesthesia and reducestress to the mouse, there is premedication with atropine (0.5 mg/kg)and xylazine (10 mg/kg) is injected subcutaneously via a 27 gaugeneedle).

To Prepare the Lesion Needle to Inject 6-OHDA:

PBS is sucked up into the line (ensure no air bubbles) with up to 200 mlin a Hamilton syringe. Syringe is placed the in the Kd Scientificsyringe pump and secured. Pump is turned the on and syringe is loadedinto the pump. Pump volume is set at 2 ml and the rate is set at 0.5ml/min. The lesion needle is positioned so that the bevelled (eye) ofthe needle is at a 45° angle from the midline and tightened to securethe needle. Pump is run and checked for fluid coming out.

Anaesthesia

3-4% isoflurane is carried by oxygen to induce anaesthesia and 1-2% formaintenance of anaesthesia. Animal is placed in small induction chamberand subjected to a few minutes of 4% isoflurane in oxygen at a flow rateof approx 1 l/min. Mouse is then placed in stereotaxic frame. Theanaesthetic tube is disconnected from induction box and isofluranereduced to 2% conc and oxygen flow rate to approx 300-400 cm/min. Tubeis connected to the nose piece attached to stereotaxic frame so thatanaesthetic gas can be constantly administered during surgery. LHS earbar is screwed at approx 3 mm. RHS ear bar is screwed in until there isno sideways head movement (about 5 mm). Front incisors are placed overthe mouthguard and put nose-guard on (3 mm below 0). Mouthguard isscrewed into place and ensured the top of the mouse's head is level(re-check this once the animal's skull is exposed).

Using a scalpel (size 4, blade 22), the scalp is cut down the centrelineof the mouse's head to expose the skull. Typically 10-15 mm. The surfaceof the skull is dried with a cotton bud. Drill hole is measured with aruler 3 mm posterior from Bregma and between 1 and 1.5 mm laterally fromthe centre line. The skull is drilled into this position until thesurface of the brain is exposed.

Toxin Loading

6-OHDA is sucked up to fill approximately 15 cm of the line and thesyringe. Fluid is ensured to be coming out of needle. The bevelled ofthe needle is aligned over the centreline where Bregma crosses, viewedby Motik dissecting microscope. The needle is ensured to be a few mmabove the skull before positioning. Frame is moved 2.9 mm in a posteriordirection and 1.1 mm (11 small notches) laterally to the right (for RHSinjections) from the starting coordinates at Bregma. Needle is moveddown until the tip of the needle just touches the surface of the brain.

Needle is lowered to the required depth of 4.6 mm and brain is allowedto settle for 2 min.

2 ml of toxin is injected into the RHS SNpc, taking 4 min. 2 min isallowed to ensure the toxin disperses. Needle is withdrawn andantiseptic applied to the wound, with suture or superglue to close.

Mouse is wrapped in a piece of tissue and placed in a warm recovery box(on a heat pad) with water bottles (50 ml tubes) warmed to 50 C.Panadol® syrup is added to mouse's water bottle.

Rotation Method

Rotation is recorded by the Computer based Rotacount system—(ColumbusInstruments, Columbus, Ohio, USA). The system comprised of 8 bowls withsensors connected to a computer which measures incremental turns (90degrees and full turns 360 degrees) either clockwise or counterclockwise. The sensors are positioned above the centre of each bowlabout 30 cm above each. Tubing and a thin empty plastic pipette areattached so that the sensor is now extended to with a few cm of the baseof the bowl.

Mice are placed individually into the eight bowls. Mice are secured tothe pipettes via a cable tie and tape. A cable tie (at least 15 cm inlength) is looped around the mouse's body (upper chest, behind frontlegs) and tightened to give a secure but not too tight hold ensuringthat the end is upright. Once tied mice are returned to their bowls fortwo minutes. Once the mice are secure, the loose ends of the cable tiecan be taped to the bottom of the pipette. This now means that anymovement by the mouse will register on the sensor. Once all mice aresecure it is then possible to start the software. Data is recorded in 5min bins. The mice are left alone in the behaviour room for 30 mins toacclimatise to their surroundings. This also gives baseline data foreach animal.

To set up the amphetamine syringes, amphetamine (Sigma) is weighed outusing SOP G8. Typically 5 mg in 4 ml of saline is enough for 12-15 micein a day. Amphetamine is injected ip at a dose of 5 mg/kg. To aid uptakeof the amphetamine, a further 300 ml of saline is taken up into eachsyringe.

Once the 30 mins has elapsed, mice are injected. Once the mice have allbeen injected, their movements are monitored for two mins as theamphetamine takes effect. The mice are left for 60 min as the computerrecords the rotations. The experiment can be stopped any time after 60mins. Rotations are recorded as CW-clockwise or CCW counter clockwise

Inclusion/Exclusion Criteria Day 3:

The animal is included if the total rotations in the 60 min postamphetamine is greater than 200 and less than 450.

The animal is excluded if less than 200 rotations are recorded in the 60mins and is no longer included in the trial. Animals failing to reachthe selection criteria are culled.

TABLE 5 6-OHDA lesion data SN cell counts Mice in Treatment - Behaviour% of SSV trial (n=)/ control or (Rotation)% SSV (SSV = 0%) mice incompound (total turns/h ± SD) (cells ± SD) expt Unlesioned mice ND284.73%  6   (6184 ± 197.07) SSV Control   0%   0% 10/26  (259.05 ±120.21) (1920.56 ± 641.47) F4076 16.54% 129.77%  9/10 (216.22 ± 86.46) (4412.80 ± 502.55) F4267 36.33% 110.44%  9/15 (30 mg kg) (164.93 ±110.90) (4041.70 ± 799.55) F4267 −23.39%  77.86% 8/10 (15 mg kg) (319.67± 171.05) (3415.96 ± 498.05) F4267  7.01% 55.79% 7/9  (5 mg kg) (240.89± 171.99) (2992.00 ± 883.84) F4268 56.04% 93.96% 9/12 (114.56 ± 71.86) (3725.04 ± 842.65) F4269 48.85% 68.91% 8/12 (132.50 ± 81.93)  (3243.93 ±724.98) F4385 49.98% 56.81% 9/17 (129.57 ± 16.26)  (3011.56 ± 502.27)F4387 24.51% 46.90% 7/12 (195.57 ± 114.48) (2821.24 ± 940.22) F449518.06% 57.22% 8/27 (212.29 ± 101.41) (3019.56 ± 318.79) F4496 36.62%95.68% 10/27  (164.20 ± 100.40) (3758.11 ± 510.98) L-DOPA 52.26%  1.73%6/9  (123.67 ± 80.90)  (1953.78 ± 280.21) Selegiline 71.13%  0.43% 6/9 (74.80 ± 57.95) (1928.89 ± 301.77)

Example 7 Parkinson's Disease In Vivo Technique: MPTP

MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) is a chemical thatis related to the opioid analgesic drugs. MPTP causes Parkinsonianside-effects. This occurs when MPTP is metabolized into MPP+, whichkills neurons in a part of the brain called the substantia nigra. MPP+interferes with mitochondria metabolism which leads to cell death andcauses the build up of free radicals, toxic molecules that contributefurther to cell destruction. MPTP has abilities to effect neuronal deathin dopaminergic cells. Such effects lead to gross depletion ofdopaminergic neurons which has severe implications on cortical controlof complex movements.

General Experimental Design Experiment 1a Determining that MPTP CausesContinued Cell Death

Control (non tg) and transgenic mice receive five intraperitonealinjections of MPTP-HCl (23 gauge needle, 20 mg\kg of free base; Sigma,St. Louis, Mo., USA) dissolved in sterile 0.9% saline at 2-h interval in1 day. Control animals receive five intraperitoneal injections of 0.9%saline. This results in a z 60% lesion of the SN. 20 C57BL6, 10 controland 10 hA53T tg are used to establish the precise dose of toxin at 2weeks. Animals are allowed to recover for 1 week, 2 weeks, 1 month, 12,months and 18 months. The animals are killed and the brains removed forhistological (stereological) and biochemical analysis.

Experiment 2 Determining Whether Test Drug Reduces MPTP Induced CellDeath

Animals are treated as described above. They are treated with test drug2 days after MPTP injection until death. Test drug is given by oralgavage at a daily dosage of 30 mg/kg. The time of killing the animals isdependant on the analysis of Experiment 1, normally one month or less.

Drug Administration

Drugs are administered by oral gavage, at a daily dosage of 30 mg/kg.

Behavioral Monitoring:

Mice are assessed at prior to killing the mice for histologicalanalysis.

Rotarod

Motor coordination and strength are assessed using the rotarod. Therotarod consists of a plastic rotating rod of 3.6 cm axial diameterpartitioned by metal disks into five sections to allow the testing ofmultiple mice simultaneously. Mice are trained on two sessions where therotation speed is ramped from 0-30 rpm over 5 min and one trainingsession where rotation is a constant 16 rpm for 5 min. Within two daysof training, animals are formally assessed on the rotarod rotating at 16rpm for a maximum of 3 min: the time to fall on this single test is therecorded data point.

Pole Test

The pole is a 700 mm long, 5 mm diameter, wooden rod. The rod issupported at its base and held vertical. The total walking distance forthe mice is 550 mm. The time taken for the mouse to descend the pole ismeasured with a maximum time of 120 s. If a mouse falls, the time isscored as 120 s.

TABLE 6 MPTP data SN cell counts Behaviour Pole % of SSV Mice in trialTreatment Test, Open field (cells +/− SD) (n =) SSV Control NAUnlesioned 30 F4267 (30 mg/kg) 285.74% (6184.72 ± 197.07 SSV 0% (2164.43± 320.79) F4267 53.94% (3331.96 ± 262.97) SSV NA 7 Day Treatment 30 CQSSV 35.2% (2180 +/− 236) CQ 44% (2725 +/− 257) SSV, F4268, Pole testbehaviour Unlesioned 50 L-DOPA (20 mg/kg) cannot be analysed 352.74%Selegiline (6184.72 ± 197.07) SSV 0% (1753 +/− 323) F4268 74.83% (3065 ±387) L-DOPA 10.39% (1935 ± 296) SSV, Pole test: Unlesioned 60 F4268Unlesioned 320.59% (30 mg/kg), 26.95% (6184.72 ± 197.07) L-DOPA (2.13 ±1.15) SSV (20 mg/kg) SSV 0% Selegiline 0% (1929.14 ± 355.82) (1 mg/kg)(2.92 ± 1.88) F4268 F468 64.00% (3163.87 ± 611.27) −25.38% L-DOPA (3.66± 1.87) 4.50% (2015.91 ± 729.87) L-DOPA Selegiline −205.29% 39.2% (8.91± 5.25) (2754.62 ± 461.23) Selegiline F4268/L-DOPA −198.93% 55.21% (8.73± 7.95) (2721.94 ± 419.73) F4268/L-DOPA −4.21% (2.77 ± 1.10) SSV ControlUnlesioned Unlesioned 50 F4486 26.95% 289.87% (30 mg/kg) (2.13 ± 1.15)(6184.72 ± 197.07) F4495 SSV SSV (30 mg/kg), 0% 0% (2133.59 ± 162.57)F4496 (2.92 ± 1.88) F4486 (30 mg/kg) F4486 24.11% (2647.97 ± 814.44)38.46% F4495 (1.81 ± 0.80) 50.33% F4495 (3207.49 ± 678.16) 18.32% F4496(2.26 ± 0.82) 69.62% F4496 (3619.04 ± 613.47) 38.61% (1.79 ± 1.13) SSVControl Unlesioned Unlesioned 50 F4095 26.95% 284.73% (30 mg/kg) (2.13 ±1.15) (6184.72 ± 197.07) F4161 SSV SSV (30 mg/kg), 0% 0% F4391 (2.92 ±1.88) (2172.12 ± 420.33) (30 mg/kg) F4095 F4095 24.25% 12.15% (2.21 ±0.89) (2436.00 ± 603.06) F4161 F4161 2.55% 28.05% (2.85 ± 1.63) (2781.33± 382.88) F4391 F4391 9.96% 25.18% (2.63 ± 1.10) (2719.11 ± 255.12) SSVControl NA Unlesioned 70 F4267 220.39% (6184.72 ± 197.07) SSV 0%(2806.26 ± 329.26) F4267 53.77% (4315.11 ± 310.21) SSV Control NAUnlesioned 70 F4269 220.39% (30 mg/kg) (6184.72 ± 197.07) SSV 0%(2806.26 ± 329.26) F4269 26.76% (3557.33 ± 556.55) SSV Control NAUnlesioned 70 F4385 220.39% (6184.72 ± 197.07) SSV 0% (2806.26 ± 329.26)F4385 31.49% (3690.00 ± 919.10) SSV Control NA Unlesioned 70 F4268220.39% (6184.72 ± 197.07) SSV 0% (2806.26 ± 329.26) F4268 40.35%(3797.33 ± 562.78) SSV Control NA Unlesioned 70 F4495 220.39% (30 mg/kg)(6184.72 ± 197.07) SSV 0% (2806.26 ± 329.26) F4495 45.45% (4081.78 ±538.77) SSV Control NA Unlesioned 70 F4496 220.39% (6184.72 ± 197.07)(30 mg/kg) SSV 0% (2806.26 ± 329.26) F4496 33.80% (3754.67 ± 619.05)

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

The invention claimed is:
 1. A method for the treatment of Parkinson'sdisease which comprises administering to a patient in need thereof aneffective amount of a compound of formula II

in which R¹ and R² are independently selected from H, optionallysubstituted C₁₋₆ alkyl and optionally substituted C₂₋₆alkynyl providedthat at least one of R¹ and R² is other than H; or R¹ and R² togetherwith the N atom to which they are attached form an optionallysubstituted 5- or 6-membered heterocycyl in which the nitrogen atom isthe only ring heteroatom and which may contain at least one further ringheteroatom selected from N and O; and R³ is methylene cyclopropyl oroptionally substituted C₃₋₆cycloalkyl; or pharmaceutically acceptablesalts thereof.
 2. The method according to claim 1 in which the compoundof Formula II is


3. A method for the treatment of Parkinson's disease which comprisesadministering to a patient in need thereof an effective amount of acompound of the formula

or a pharmaceutically acceptable salt thereof in which R¹ and R² takentogether with the nitrogen atom to which they are attached form anoptionally substituted 5- or 6-membered heterocyclyl with the nitrogenatom being a ring heteroatom, wherein said heterocyclyl may contain atleast one further ring heteroatom selected from N and O and R³ isselected from optionally substituted C₁₋₆ alkyl and optionallysubstituted C₃₋₆ cycloalkyl.
 4. The method according to claim 3 in whichthe compound is


5. A method for the treatment of Parkinsons Disease comprisingadministering to a patient in need thereof the compound of formula II:

in which R¹ and R² are independently selected from H, optionallysubstituted C₁₋₆ alkyl and optionally substituted C₂₋₆alkynyl providedthat at least one of R¹ and R² is other than H; or R¹ and R² togetherwith the N atom to which they are attached form an optionallysubstituted 5- or 6-membered heterocyclyl which may contain at least onefurther ring heteroatom selected from N and O; and R³ is methylenecyclopropyl, or pharmaceutically acceptable salts thereof.
 6. A methodof treating Parkinsons Disease comprising administering to a patient inneed thereof, a compound of Formula II

wherein, in said compound, R¹ is C₁₋₄alkyl; R² is selected fromC₁₋₄alkyl optionally substituted with optionally substituted aryl orC₂₋₆alkynyl; or R¹ and R² together with the N atom to which they areattached form an optionally substituted 5- or 6-membered heterocyclylwhich may contain at least one further ring heteroatom selected from Nand O; and R³ is C₁₋₄alkyl or pharmaceutically acceptable salts thereof.7. A method of treating Parkinsons Disease comprising administering to apatient in need thereof, a compound selected from the following:

or pharmaceutically acceptable salts thereof.
 8. A method of treatingParkinsons Disease comprising administering to a patient in need thereofthe compound

in which R¹ _(c) is selected from H and C₁₋₄alkyl; R² _(c) is selectedfrom C₁₋₄alkyl, C₂₋₆cycloalkyl methyl substituted with optionallysubstituted aryl and C₂₋₄alkynyl; or R¹ _(c) and R² _(c) together withthe N atom to which they are attached form an optionally substituted 5-or 6-membered heterocyclyl which may contain at least one furtherheteroatom selected from N and O; and R³ _(c) is selected fromoptionally substituted C₃₋₆cycloalkyl, optionally substituted aryl andoptionally substituted 5- or 6-membered heterocyclyl containing at leastone heteroatom selected from N and O; or pharmaceutically acceptablesalts thereof.
 9. The method according to claim 8 in which the compoundis selected from the following:

or pharmaceutically acceptable salts thereof.